Recombinant mycobacteria

ABSTRACT

Recombinant mycobacterial vaccine vehicles capable of expressing DNA of interest which encodes at least one protein antigen for at least one pathogen against which an immune response is desired and which can be incorporated into the mycobacteria or stably integrated into the mycobacterial genome. The vaccine vehicles are useful for administration to mammalian hosts for purposes of immunization. A recombinant vector which replicates in E. coli but not in mycobacteria is also disclosed. The recombinant vector includes 1) a mycobacterial gene or portions thereof, necessary for recombination with homologous sequences in the genome of mycobacteria transformed with the recombinant plasmid; 2) all or a portion of a gene which encodes a polypeptide or protein whose expression is desired in mycobacteria transformed with the recombinant plasmid; 3) DNA sequences necessary for replication and selection in E. coli; and 4) DNA sequences necessary for selection in mycobacteria (e.g., drug resistance). The present invention also relates to two types of recombinant vectors useful in introducing DNA of interest into mycobacteria, where it is expressed. One type of vector is a recombinant plasmid capable of replicating as a plasmid in E. coli and of lysogenizing a mycobacterial host. The other type of vector is a recombinant plasmid which can be introduced into mycobacteria, where it is stably maintained extrachromosomally.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 07/361,944,now U.S. Pat. No. 5,504,005, filed Jun. 5, 1989, entitled "RecombinantMycobacterial Vaccine", which is a continuation-in-part of applicationSer. No. 07/223,089, now abandoned, filed Jul. 22, 1988, entitled"Stable Expression of Cloned Genes in Mycobacteria Using Phage andPlasmid Vectors" and of application Ser. No. 07/216,390, now abandoned,filed Jul. 7, 1988, entitled "Recombinant Mycobacteria Having DNA ofInterest Stably Integrated Into Genomic DNA, which arecontination-in-part applications of application Ser. No. 07/163,546, nowabandoned, filed Mar. 3, 1988, entitled "Recombinant MycobacterialVaccine", which is a continuation-in-part of application Ser. No.07/020,451, now abandoned, filed Mar. 2, 1987, entitled "RecombinantMycobacterial Vaccine." The teachings of these four related applicationsare incorporated herein by reference.

FUNDING

Work described herein was supported by funding from the World HealthOrganization, Albert Einstein College of Medicine of Yeshiva University,The Whitehead Institute for Biomedical Research, the National Institutesof Health, and the Rockefellar Foundation.

BACKGROUND Immunization

Immunity to a foreign antigen (e.g., a pathogen or toxin) can beprovided by passive transfer or active induction. In the former case,antibodies against the foreign protein pathogen are injected into anindividual, with the result that short-term protection is provided. Inthe latter case, injection of a harmless (innocuous) form of thepathogen, a component of the pathogen, or a modified form of the toxin(i.e., a toxoid) stimulates the individual's immune system, conferringlong-term protection.

Active immunity can be induced, provided an individual's immune systemis competent, by using an appropriate antigen to stimulate the immunesystem. For example, immunization (vaccination) with an innocuous orattenuated form of the pathogen in this manner results in an immediateimmune response, as well as immunological "memory", thus conferringlong-term protection as well. In general, vaccines include inactivated,nonpathogenic or attenuated forms of a pathogen or infectious agent,which include antigenic determinants of the pathogen and thus elicit animmune response. Similarly, toxins, which are antigenic substancesproduced by microorganisms, plants and animals, can be converted totoxoids; that is, they can be modified to destroy their toxic propertiesbut retain their antigenicity and, as a result, their ability tostimulate production of antitoxin antibodies and produce activeimmunity. Such toxoids can be used for vaccines against the toxin.

In both cases--that involving stimulation of an immune response byadministration of an altered form of an infectious pathogen and thatinvolving administration of a toxoid--presently-available procedures aregenerally effective, but side effects and deaths resulting from thevaccination are known to occur.

Safer vaccines are now being developed through application of betterknowledge of the antigenic determinants of a pathogen and of geneticengineering/recombinant DNA techniques. For example, it is possible tomake a polypeptide (e.g. by chemical synthesis or expression of DNAencoding the polypeptide of interest) which is a component (e.g., anantigenic determinant) of a protein antigen known to elicit an immuneresponse. Administration of the polypeptide to a host is followed by animmune response by the host to the antigenic determinant. Use of such apolypeptide is not accompanied by the risk of infection whichaccompanies use of live or attenuated vaccines.

Immunization (administration of a vaccine) is a common and widespreadprocedure and the vaccine used can be essentially "any preparationintended for active immunological prophylaxis", including preparationsof killed microbes of virulent strains, living microbes of attenuatedstrains, and microbial, fungal, plant, protozoal or metazoan derivativesor products. Stedman's Illustrated Medical Dictionary (24th edition),Williams & Wilkins, Baltimore, p. 1526 (1982). In many cases, vaccinesmust be administered more than once in order to induce effectiveprotection; for example, known anti-toxin vaccines must be given inmultiple doses.

Childhood vaccination is commonplace and generally successful indeveloped countries, where there is ready access to health services andmultiple immunizations (e.g. immunization against multiple pathogens andserial or multiple immunizations against a single pathogen) arepossible. In the developing world, vaccination is far less common andfar more problematic. For example, only about 20 percent of the 100million children born in the developing world each year are vaccinatedagainst diphtheria, pertussis, tetanus, measles, poliomyelitis andtuberculosis. It is estimated that each year, 5 million children in thedeveloping world die and another 5 million children are physically ormentally disabled by these diseases, which could be prevented ifadequate immunization were possible. Availability of effective vaccineswhich can confer long-term immunity with a single administration would,of course, be valuable in both developed and developing countries.

Vaccination of adults is also helpful in preventing many diseases inadults and, as is the case with children, in developing countries mayprove to be difficult to carry out, particularly if multipleimmunizations are necessary. Diseases such as leprosy, malaria,tuberculosis, and poliomyelitis, among others, have a high incidenceamong adults in Africa, Asia and Latin America and are the causes ofthousands of deaths annually.

Much effort has been expended in developing vaccines against majordiseases and, recently, consideration has been given to recombinantvaccine vehicles (e.g., genetically engineered viruses) to expressforeign genes. For example, recombinant vaccinia virus, in which viralantigens are inserted into vaccinia virus--has been developed. Forexample, hepatitis B genes, influenza virus genes or DNA encoding rabiesvirus antigen have been spliced into vaccinia virus DNA in efforts tomake vaccines. Panicali, D. et. al., Proceedings of the National Academyof Sciences, USA, 80: 5364-5368 (1983); Orr, T., Genetic EngineeringNews, p. 17, (March 1985); Paoletti, E. and D. Panicali, U.S. Pat. No.4,603,112.

It is widely agreed, however, that such recombinant vaccinia virus wouldhave at least two important drawbacks as a vaccine. First, there is asignificant mortality and morbidity (1:100,000) associated with vacciniavirus, which is untreatable. Second, vaccination with recombinantvaccinia of individuals previously exposed to vaccinia virus has oftenfailed to produce satisfactory immunization levels. Fenner, F., NewApproaches to Vaccine Development, R. Bell and G. Torrigiani (ed.),Schwabe & Co., p. 187 (1984).

To date, vaccines have been developed which, although effective in manyinstances in inducing immunity against a given pathogen, must beadministered more than once and may be unable to provide protection, ona long-term basis, against a pathogen. In addition, in many cases (e.g.,leprosy, malaria, etc.), an effective vaccine has yet to be developed.

Mycobacteria

Mycobacteria represent major pathogens of man and animals. For example,tuberculosis is generally caused in humans by Mycobacterium (M.)tuberculosis and in cattle by Mycobacterium (M.) bovis (which can betransmitted to humans and other animals, in whom it causestuberculosis). Tuberculosis remains widespread and is an importantpublic health problem, particularly in developing countries. It isestimated that there are approximately 10 million cases of tuberculosisworld-wide, with an annual mortality of 3 million. Joint InternationalUnion Against Tuberculosis and World Health Organization Study Group,Tubercle, 63:157-169 (1982).

Leprosy, which is caused by M. leprae, afflicts over 10 million people,primarily in developing countries. Bloom, B. R. and T. Godal, Review ofInfectious Diseases, 5:657-679 (1984). M. tuberculosis and mycobacteriaof the avium-intracellulare-scrofulaceum (MAIS) group represent majoropportunistic pathogens of patients with acquired immunodeficiencydisease (AIDS). Centers for Disease Control, Morbidity and MortalityWeekly Report, 34:774 (1986). M. pseudotuberculosis is a major pathogenof cattle.

On the other hand, Bacille Calmette-Guerin (BCG), an avirulent strain ofM. bovis, is the most widely used human vaccine in the world and hasbeen used as a live vaccine for more than 50 years. In the past 35years, it has been administered to over 2.5 billion people, withremarkably few adverse effects (e.g., estimated mortality of60/billion). BCG has been found in numerous studies to have protectiveefficacy against tuberculosis. Recently, however, it was found not to beeffective in preventing pulmonary tuberculosis in Southern India.Tuberculosis Prevention Trial, Madras, Indian Journal of MedicalResearch, 72 (suppl.):1-74 (1980).

Thus, although there are numerous vaccines available, including BCG,many are limited in value because they induce a limited immune response,must be given in multiple doses and/or have adverse side effects. Inother cases (e.g., leprosy, malaria), a vaccine is simply unavailable.It would be of great value if a vaccine against a pathogen or pathogensof concern were available which provided long-term stimulation ofimmunity in recipients sufficient to provide protection against thepathogen(s) without adverse effects.

DISCLOSURE OF THE INVENTION

The present invention relates to genetically recombinant (geneticallyengineered) cultivable mycobacteria which express DNA of interest whichhas been incorporated into the mycobacteria, in which it is present inthe mycobacterial genome or extrachromosomally, using geneticengineering techniques; to vectors useful for the introduction of DNA ofinterest into mycobacteria; to methods of introducing DNA intomycobacteria and to methods of incorporating or integrating DNA stablyinto the mycobacterial genome to produce genetically recombinantmycobacteria. It further relates to a method of transferring geneticmaterial between different genera of microorganisms by means ofgenetically engineered shuttle vectors, which are shuttle phasmids orshuttle plasmids. These shuttle vectors, which are also the subject ofthe present invention, are useful for the transfer of genetic materialbetween different genera of microorganisms and introduction of DNA ofinterest into mycobacteria.

Recombinant DNA vectors of the present invention are of two types: atemperate shuttle plasmid and a bacterial-mycobacterial shuttle plasmid(e.g., E. coli mycobacterial shuttle plasmid). Each type of recombinantvector can be used to introduce DNA of interest stably intomycobacteria, in which the DNA can then be expressed. In the case of thetemperate shuttle plasmid, which includes DNA of interest, stableintegration into the mycobacterial chromosomal or genomic DNA occurs viasite specific integration. The DNA of interest is replicated as part ofthe chromosomal DNA. In the case of the bacterial-mycobacterial shuttleplasmid, which includes DNA of interest, the DNA of interest is stablymaintained extrachromosomally as a plasmid (as a component of theplasmid). Expression of the DNA of interest occurs extrachromosomally asa plasmid (e.g., episomally). For example, a gene or genes of interestis/are cloned into a bacterial-mycobacterial plasmid and introduced intoa cultivable mycobacterium, where it undergoes episomal replication(extrachromosomal replication). As a result of the work describedherein, promoters which will express in mycobacteria have been defined;for example, the promoter expressing kanamycin resistance, the promoterexpressing chloramphenicol resistance and the cI promoter have beenshown to express in mycobacteria.

The recombinant vectors of the present invention are useful in themethod of the present invention, by which genetic material can betransferred between different genera of microorganisms (e.g., betweenbacteria and mycobacteria). They have made it possible to introduce intomycobacteria, such as Mycobacterium smegmatis (M. smegmatis) andMycobacterium bovis-BCG (BCG), DNA from another source (e.g., DNA from asource other than the mycobacterium into which the DNA is beingincorporated--for example, M. smegmatis or BCG). The DNA from anothersource is referred to herein as DNA of interest. Such DNA of interestcan be of any origin and is: 1) DNA which is all or a portion of a geneor genes encoding protein(s) or polypeptide(s) of interest; 2) DNAencoding a selectable marker or markers; or 3) DNA encoding both aselectable marker or markers and at least one protein or polypeptide ofinterest. The proteins or polypeptides of interest can be, for example,proteins or polypeptides against which an immune response is desired(antigen(s) of interest), enzymes, lymphokines, immunopotentiators, andreporter molecules of interest in a diagnostic context.

DNA of interest can be integrated or incorporated into the mycobacterialgenome and is referred to as integrated DNA or integrated DNA ofinterest. As a result, DNA of interest can be introduced stably into andexpressed in mycobacteria (i.e., production of foreign proteins iscarried out from the DNA of interest present in the mycobacteria).Alternatively, DNA of interest is integrated into mycobacterial DNA,through the method of the present invention, as a result of homologousrecombination. According to the method of the present invention, arecombinant plasmid is used for introduction of DNA of interest intomycobacterial cells and for stable integration of the DNA into themycobacterial genome. The recombinant plasmid used includes: 1)mycobacterial sequences (referred to as plasmid-borne mycobacterialsequences) necessary for homologous recombination to occur (betweenplasmid-borne mycobacterial sequences and sequences in the mycobacterialgenome); 2) DNA sequences necessary for replication and selection in E.coli; and 3) DNA of interest (e.g., DNA encoding a selectable marker andDNA encoding a protein or polypeptide of interest). The recombinantplasmid is introduced, using known techniques, into mycobacterial cells.The mycobacterial sequences in the plasmid can be identical to thosepresent in the mycobacterial genome or sufficiently similar to thosepresent in the mycobacterial genome to make homologous recombinationpossible. "Recognition" of homology of sequences present in theplasmid-borne mycobacterial DNA and identical of sufficiently similarsequences present in the mycobacterial genome results in crossoverbetween the homologous regions of the incoming (plasmid-borne)mycobacterial DNA and the genomic mycobacterial DNA and integration ofthe recombinant plasmid into the mycobacterial genome. Integrationoccurs at a selected site in the mycobacterial genome which isnon-essential, (i.e., not essential for mycobacterial replication).Integration of the homologous plasmid sequences is accompanied byintegration of the DNA of interest into the mycobacterial genome.

The present invention further relates to recombinant mycobacteria whichexpress DNA of interest which has been integrated into the mycobacterialDNA or which is maintained extrachromosomally as a plasmid. Suchrecombinant mycobacteria can be produced by introducing DNA of interestinto any appropriate mycobacterium, such as M. smegmatis, M. bovis-BCG,M. avium, M. phlei, M. fortuitum, M. lufu, M. paratuberculosis, M.habana, M. scrofulaceum and M. intracellulare. In recombinantmycobacteria in which DNA of interest is integrated into genomic DNA,the DNA of interest is present in such a manner that 1) a mycobacterialgene is replaced (i.e., is no longer present in the mycobacterialgenome) or 2) the DNA of interest is inserted into a mycobacterial gene,with the result a) that the mycobacterial gene is left intact andfunctional or b) that the mycobacterial gene is disrupted and renderednonfunctional.

The resulting genetically recombinant mycobacteria (e.g., recombinantBCG, recombinant M. smegmatis) are particularly useful as vehicles bywhich the DNA of interest can be expressed. These are referred to asgenetically recombinant mycobacteria or mycobacterial expressionvehicles. Such vehicles can be used, for example, as vaccine vehicleswhich express a polypeptide or a protein of interest (or more than onepolypeptide or protein), such as an antigen or antigens, for one or morepathogens of interest. The recombinant mycobacteria can also be used asa vehicle for expression of immunopotentiators, enzymes, pharmacologicagents and antitumor agents; for expression of a polypeptide or aprotein useful in producing an anti-fertility vaccine vehicle; or forexpression of stress proteins, which can be administered to evoke animmune response or to induce tolerance in an autoimmune disease (e.g.,rheumatoid arthritis). Recombinant mycobacteria can, for example,express protein(s) or polypeptide(s) which are growth inhibitors or arecytocidal for tumor cells (e.g., interferon α, β or γ; interleukins 1-7,tumor necrosis factor (TNF) α or β) and, thus, provide the basis for anew strategy for treating certain human cancers (e.g., bladder cancer,melanomas). Pathogens of interest include any virus, microorganism, orother organism or substance (e.g., a toxin or toxoid) which causesdisease. The present invention also relates to methods of vaccinating ahost with the recombinant mycobacterium to elicit protective immunity inthe host. The recombinant vaccine can be used to produce humoralantibody immunity, cellular immunity (including helper and cytotoxicimmunity) and/or mucosal or secretory immunity. In addition, the presentinvention relates to use of the antigens expressed by the recombinantcultivable mycobacterium as vaccines or as diagnostic reagents.

The vaccine of the subject invention has important advantages overpresently-available vaccines. First, mycobacteria have adjuvantproperties among the best currently known and, thus, stimulate arecipient's immune system to respond to other antigens with greateffectiveness. This is a articularly valuable aspect of the vaccinebecause it induces cell-mediated immunity and will, thus, be especiallyuseful in providing immunity against pathogens in cases wherecell-mediated immunity appears to be critical for resistance. Second,the mycobacterium stimulates long-term memory or immunity. As a result,a single (one-time) inoculation can be used to produce long-termsensitization to protein antigens. Using the vaccine vehicle of thepresent invention, it is possible to prime long-lasting T cell memory,which stimulates secondary antibody responses neutralizing to theinfectious agent or the toxin. This is useful, for example, againsttetanus and diphtheria toxins, pertusis, malaria, influenza, herpesviruses and snake venoms.

BCG in particular has important advantages as a vaccine vehicle inthat: 1) it is the only childhood vaccine currently given at birth; 2)in the past 40 years, it has had a very low incidence of adverseeffects, when given as a vaccine against tuberculosis; and 3) it can beused repeatedly in an individual (e.g., in multiple forms).

A further advantage of BCG in particular, as well as mycobacteria ingeneral, is the large size of its genome (approximately 3×10⁶ bp inlength). Because the genome is large, it is able to accommodate a largeamount of DNA from another source (i.e., DNA of interest) and, thus, canbe used to make a multi-vaccine vehicle (i.e., one carrying DNA ofinterest encoding protective antigens for more than one pathogen).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of transfection of Mycobacterium smegmatisspheroplasts with mycobacteriophage D29 DNA.

FIG. 2 is a schematic representation of the construction of the shuttleplasmid, phAE1.

FIGS. 3A and 3B shows results of assessment of shuttle plasmid phAE1.

FIG. 3A shows agarose gel of mycobacteriophage TM4 DNA and shuttleplasmid phAE1 DNAs digested with KpnI. Lane 1 contains lambda DNAdigested with Hind III; lanes 2 and 3 contain TM4 DNA that was unligated(lane 2) or ligated (lane 3) prior to digestion cut with KpnI; lanes 4and 5 contain phAE1 DNA isolated from phage particles propagated on M.smegmatis (lane 4) and phAE1 isolated from E. coli cells as a plasmid(lane 5). Note that the arrows point to the 2.1 Kb and the 1.8 Kbfragments that form a 3.9 Kb fragment when ligated at the cohesive ends.

FIG. 3B shows results of a Southern blot analysis of plasmid phAE1,using pHC79 as a probe (panel B). The autoradiograph of FIG. 3A is shownafter blotting onto a Biotrans nylon membrane (ICN) and probing withpHC79 DNA that had been nick-translated with ³² P-dCTP.

FIGS. 4A-4C shows replication of phAE1 on BCG. It compares lysis of theGlaxo vaccine strain of BCG by DS6A, which is a mycobacteriophage knownto plaque on M. tuberculosis and BCG, but not on other mycobacteria;phage 33D, known to plaque on M. smegmatis and not BCG; and phage TM4,which plaques on both species.

FIG. 4A shows lysis of BCG by the phages. Titres of phage (pfu/ml) usedat 10⁻¹ dilution were: DS6a, 2×10⁶ on M. tuberculosis, H37Ra; 33D, 2×10⁶on M. smegmatis, mc² 6; TM4, 3×10⁸ on mc₂ 6; and phAE1, 3×10⁸ on mc² 6.Dilutions of phages (5 ul) were spotted on a soft agar overlaycontaining 10⁸ BCG cells. Resulting lysis was photographed afterincubation for 10 days at 37° C.

FIG. 4B shows the presence of cosmid DNA in phAE1. Plaque lifts on theseplates were carried out as described below and hybridized with ³²P-labelled pHC79 DNA; this was followed by autoradiography.

FIG. 4C is an electron micrograph of shuttle plasmid phAE1 phageparticles. Phage particles that had been purified on CsCl gradients wereplaced on carbon coated, Parloidon-coated grids, blotted and washed withone drop of 1% phosphotungstic acid. Electron micrographs were takenusing a JEOL 1200EX electron microscope at 80 kV, 30,000×.

FIG. 5 shows integration of mycobacteriophage L1 and L1-shuttle plasmidDNA into the M. smegmatis chromosome. DNAs from phage L1 and L1-shuttlephasmids and chromosomal DNAs from corresponding lysogens were digestedwith BamHI and electrophoresed in agarose. Panel A shows an ethidiumbromide stained gel. Panel B shows the autoradiograph of the Southernanalysis of this gel probed with ³² P-labelled phage L1 DNA. Thefollowing are shown in the lane indicated: phage DNA from the parentphage L1 (lane 2), from shuttle plasmid phAE15 (lane 4) and from shuttleplasmid phAE19 containing the aph gene (lane 6); bacterial chromosomalDNA from the parent M. smegmatis strain (lane 1), from that strainlysogenized with L1 (lane 3), with phAE15 (lane 5), and with phAE19(lane 7). L1, phAE15, and phAE19 have integrated site-specificallywithin the chromosome of their respective lysogens (Panel B, lanes 3, 5& 7), as evidenced by the predominant loss of a single 6.7 kb bandpresent in each phage (note square in L1, lane 2) and the appearance oftwo new bands, 9.0 kb and 1.7 kb, in each lysogen (circles).

FIG. 6 is a schematic representation of the use of temperate shuttlephasmids as cloning vectors to stably introduce DNA of interest into themycobacterial chromosome. DNA of interest (designated GENE X) can beinserted into unique restriction sites in shuttle plasmid DNA andsubsequently introduced into mycobacteria. In mycobacteria, the shuttleplasmid, carrying the DNA of interest, can lysogenize and be maintainedstably as a prophage.

FIG. 7 shows expression of kanamycin-resistance by lysogeny using thetemperate shuttle plasmid phAE19. Colonies appeared where phAE19lysogenized mc² 6 cells, thus demonstrating expression ofkanamycin-resistance. In multiple experiments, kanamycin-resistancecolonies were not observed from either spontaneous mutants of mc² 6cells or mc² 6 cells lysogenized with phAE15.

FIG. 8 is a schematic representation of the overall strategy used togenerate a library of hybrid plasmid molecules consisting of an E. coliplasmid, pIJ666, that contains marker genes conferring resistance toneomycin/kanamycin (neo) and chloramphenicol (cat), inserted at randomsites around the pAL5000 genome.

FIG. 9 shows results of agarose gel electrophoretic analysis of DNA frompIJ666::pAL5000 recombinant shuttle plasmids isolated from 3 independentpools of M. smegmatis transformants (lanes 1, 2, 3). Following separatetransformations of each of these plasmid pools into E. coli strainχ2338, unique plasmids were isolated from single purified transformants,designated pYUP13, pYUP14 and pYUP15, and are shown in lanes 5, 6, and7, respectively. Lane 4 contains the M. fortuitum plasmid, pAL5000, andlane 8 contains the library of pIJ666::pAL5000 recombinants. The size ofthe shuttle plasmids isolated from either M. smegmatis or E. coli isidentical to the size of the recombinant library, indicating stabilityof the construct.

FIGS. 10A & 10B show transformation of BCG with shuttle plasmid DNA.Panel A showns kanamycin-resistant BCG colonies that arose afterelectroporation of BCG cells with shuttle plasmid DNA; Panel B showskanamycin-resistant BCG colonies that arose after electroporation of BCGcells without shuttle plasmid DNA.

FIG. 11 is a schematic representation of the construction of arecombinant plasmid in which there is a Kan insertion in the PyrF geneof the plasmid vector pUC19.

FIGS. 12A and 12B is a schematic representation of transformation ofmycobacterial cells with the pUC19 recombinant plasmid in which the PyrFgene contains a Kan insertion. FIG. 12A is a schematic representation ofselection, using growth on kanamycin-containing medium, of mycobacterialcells in which the PyrF gene containing the Kan gene is present. FIG.12B is a schematic representation of selection, using growth onfluoro-orotic acid-containing medium, of mycobacterial cells having thePyrF gene containing the Kan gene integrated into genomic DNA.

FIG. 13 is a schematic representation of the integration of Kan and DNAencoding a selected antigen (designated Fan) into mycobacterial DNA.

FIG. 14 is a schematic representation of replacement of themycobacterial PyrF gene with a gene encoding kanamycin resistance.

FIG. 15 is a schematic representation of integration of a PyrF gene andDNA of interest into a recombinant mycobacterium produced as representedin FIG. 14.

FIG. 16 is a schematic representation of the use of an expressioncassette to control expression of DNA of interest integrated into amycobacterial genome.

FIG. 17 shows results of Western blot analysis showing expression of theM. leprae gene encoding stress-induced 65 kD antigen in M. smegmatis andBCG.

DETAILED DESCRIPTION OF THE INVENTION

Mycobacterium bovis-BCG (BCG or M. bovis-BCG) is an avirulent M. bovisderivative which is widely used throughout the world and is commonlyused to provide protection against tuberculosis, although itseffectiveness has recently been called into question. Mycobacteriumsmegmatis is a nonpathogenic bacillus which shares antigenic andadjuvant properties with BCG. Both are also reasonably easy to grow inculture.

Because both mycobacteria have excellent adjuvant activity for inductionof cell-mediated immunity, stimulate long-term memory (immunity) andhave a low mortality associated with their use, they are excellentcandidates as recombinant vaccines. That is, they are excellentcandidates for use as vehicles (vaccine vehicles) into which geneticmaterial (DNA) of interest (DNA from a source other than themycobacterium into which it is being introduced) can be inserted andsubsequently expressed.

DNA of interest can be of any origin and is: 1) DNA which is all or aportion of a gene or genes encoding protein(s) or polypeptide(s) ofinterest; 2) DNA encoding a selectable marker or markers; or 3) DNAencoding both a selectable marker (or selectable markers) at least oneprotein or polypeptide of interest. The term polypeptide of interest,used herein, includes all or a portion of a protein to be expressed.Such DNA of interest is expressed in the genetically recombinantmycobacteria, in which it is present in (integrated into) themycobacterial genome or is present extrachromosomally. Incorporated DNA,as defined herein, includes DNA present in chromosomal DNA or present inmycobacteria extrachromosomally (episomally). DNA is incorporated bymeans of a shuttle plasmid or shuttle plasmid, resulting in integrationinto mycobacterial chromosomal or genomic DNA or the presence of DNA ofinterest episomally (extrachromosomally). Integration of DNA of interestcan occur by homologous or nonhomologous recombination, such assite-specific recombination of a phage-encoded system or recombinationmediated by a transposable element.

Until the present time it has not been possible to transform amycobacterium through the use of plasmid DNA. Further, until now, it hasnot been possible to produce recombinant mycobacterial vaccine vehiclesin which DNA encoding a polypeptide or protein such as one against whichan immune response is desired, is stably integrated, at selected sitesand in selected orientations, in genomic DNA.

A principal objective of work on the development of a recombinantmycobacterium to be used as an expression vehicle or a vaccine vechicleis the introduction into the mycobacterium of DNA vectors that directthe expression of DNA encoding a product or products, such as a proteinor polypeptide, important for protection against one or more pathogens.It is now possible, using the method and the shuttle or plasmid vectorof the present invention, to incorporate DNA of interest into acultivable mycobacterium (e.g., into the mycobacterial genome or intothe mycobacterium in such a manner that it is expressedextrachomosomally).

The shuttle plasmid vector of the present invention is unique in that itreplicates as a plasmid in bacteria and as a phage in mycobacteria. In aparticular embodiment, the shuttle plasmid vector, which is referred toas a shuttle plasmid, includes two species of specific cohesive end (orcos sites): one for lambda phage, which functions in E. coli; and onefor mycobacteria (e.g., the mycobacteriophage TM), which functions inmycobacteria. That is, it contains two sets of cohesive ends. Because itcontains one set for lambda and one for mycobacteria, it can beincorporated into both. The presence of the lambda COS sequence alsomakes it possible to use the efficient technique of cosmid cloning,which utilizes the lambda in vitro packaging system for efficientcloning of large DNA molecules into E. coli. Further, the shuttle vectorhas a unique EcoRI site into which antigen-encoding DNA can be inserted.Thus, the shuttle vectors have made it possible to develop atransfection system which permits introduction of recombinant DNAmolecules into mycobacteria.

There are several means by which genetic material of interest can beincorporated into mycobacteria to produce recombinant mycobacteria ofthe present invention. For example, DNA of interest can be stablyintroduced (e.g., integrated into the mycobacterial chromosome) intomycobacterial cells by cloning into a shuttle plasmid, particularly atemperate shuttle plasmid (e.g., a phage capable of lysogenizing acell). Introduction of DNA of interest in this manner results inintegration of the DNA into the mycobacterial chromosome.

For example, an E. coli cosmid was introduced into the temperatemycobacteriophage L1, producing shuttle phasmids capable of replicatingas plasmids in E. coli or lysogenizing the mycobacterial host. Thesetemperate shuttle phasmids form turbid plaques on M. smegmatis and, uponlysogenization, confer resistance to superinfection and integrate withinthe mycobacterial chromosome. When an L1-shuttle plasmid containing acloned gene conferring kanamycin-resistance in E. coli was introducedinto M. smegmatis, stable kanamycin-resistant colonies (i.e., lysogens)were obtained.

Alternatively, a plasmid vector can be used to introduce DNA of interestinto mycobacteria, in which the DNA is expressed extrachromosomally. Forexample, the shuttle plasmid M. fortuitum::E. coli hybrid plasmids wereconstructed from mycobacterial and E. coli replicons which containkanamycin- and chloramphenicol-resistance genes. When introduced into M.smegmatis or BCG by electroporation, these shuttle plasmids conferredstable kanamycin- and chloramphenicol-resistance upon transformants.Thus, the vectors have made it possible to develop a transfection systemwhich permits introduction of recombinant DNA molecules intomycobacteria.

It is also possible to introduce DNA of interest and cause it tointegrate into host chromosomes without a phage. For example, this canbe accomplished by homologous recombination, site specific recombinationor nonhomologous recombination (e.g., by means of a transposon, whichresults in random insertion into host chromosomal material). Homologousrecombination has been used, as described below, to integrate DNA ofinterest (e.g., kanamycin-resistance gene, 65 KD M. leprae gene).

In order to successfully introduce DNA of interest into a mycobacteriumor into the mycobacterial genome by means of the shuttle vector orplasmid vector of the present invention or by homologous recombination,the following general approaches were followed. Although it is describedin terms of M. smegmatis and M. bovis-BCG, it is to be understood thatit can also be used to introduce DNA of interest into other mycobacteriaand that these other genetically recombinant mycobacteria can also beexpression or vaccine vehicles. Such other mycobacteria include: M.smegmatis, M. bovis-BCG, M. avium, M. phlei, M. fortuitum, M. lufu, M.paratuberculosis, M. habana, M. scrofulaceum, and M. intracellulare. Inthe case of slow growing mycobacteria (e.g., M. bovis-BCG and M.tuberculosis) to be used as vaccine vehicles, it is particularlyvaluable to go through (i.e., introduce DNA encoding an antigen orantigens of interest into) M. smegmatis and, subsequently, into M.bovis-BCG.

Development of a shuttle vector to transfer DNA into mycobacteria

Transfection of mycobacteriophage DNA into M. smegmatis

To develop a system that permits manipulation of DNA in mycobacteria, itwas first necessary to develop an efficient means of transferring DNAinto the bacillus. The technology used was a modification of thatdescribed by Okanishi and Hopwood in relation to the preparation ofspheroplasts for Streptomyces. Streptomyces, like mycobacteria, areActinomycetales. Okanishi, M. et al., Microbiology, 80: 389-400 (1974);Hopwood, D. A. and H. M. Wright, Molecular Genetics, 162: 307-317(1978). The modified technique was used in combination with the additionof polyethylene glycol to facilitate entry of DNA molecules intobacterial spheroplasts.

Because of the unavailability of useful selectable antibiotic resistancemarkers in plasmids for transforming mycobacteria, the system chosen toevaluate optimum conditions for DNA transfer into mycobacteria was thetransfection of DNA from lytic mycobacteriophages. Two advantages ofsuch a system are that results obtained were quantitative and readilyvisualized as plaques within 24 hours.

Transfection of mycobacteriophage DNA into M. smegmatis is described indetail in Example 1. Briefly, DNA was initially introduced intomycobacteria having all or a portion of the cell walls removed (i.e.,protoplasts or spheroplasts), using polyethylene glycol. Transfectionexperiments were initiated with DNA from mycobacteriophage D29, whichpropagates on a wide variety of mycobacteria and forms large clearplaques on M. smegmatis. Plate lysates of D29 phage prepared on M.smegmatis consistently yielded greater than 10¹¹ pfu (plaque formingunits) per ml of lysate. The harvested phages were twice purified onCsCl equilibrium gradients; they banded at an equilibrium buoyantdensity of 1.51. Phage DNA was extracted by proteinase K treatment andphenol-chloroform extraction. Restriction analysis of ligated andunligated D29 DNA demonstrated that the phage genomic DNA was doublestranded, 50 kb in size, and possessed cohesive ends.

The results of transfection of M. smegmatis spheroplasts bymycobacteriophage D29 DNA are illustrated in FIG. 1. Efficiencies of 10³to 10⁴ pfu per ug D29 DNA were obtained, thus demonstrating the firstefficient transfection system for mycobacteria. That these plaques werethe result of transfection of M. smegmatis spheroplasts was demonstratedby the following: (i) transfection was abolished by DNase; (ii) osmoticshock of treated cells prevented productive transfection; and (iii)spheroplasts derived from a D29 phage-resistant mutant of M. smegmatiswere transfected at frequencies comparable to the parent strain. Furtherrefinement of these techniques made it possible to obtain frequenciesgreater than 10⁵ pfu per ug of D29 DNA.

Introduction of DNA of interest into mycobacteria

A vector that would permit both the manipulation and amplification ofmycobacterial DNA constructs in E. coli, and subsequent transfer intoand replication in mycobacteria was developed. In particular, it washighly desirable to have the capability of introducing DNA of interestinto fast-growing non-pathogenic mycobacterium (e.g., M. smegmatis), aswell as into slow-growing mycobacteria (e.g., M. bovis-BCG and M.tuberculosis). Although plasmids have been found in some mycobacterialstrains within the MAIS complex and in M. fortuitum, none have yet beendescribed which replicate within M. smegmatis, M. bovis-BCG, or M.tuberculosis. With one exception, none of these plasmid possessselectable markers. Crawford, J. T. and J. H. Bates Infections andImmunity, 24: 979-981 (1979); Mizuguchi, Y. et al., Journal ofBacteriology, 146: 656-659 (1981); Meissner, P. S. and J. O. Falkinham,Journal of Bacteriology, 157: 669-672 (1984). In contrast, a variety ofphages that replicate in M. smegmatis, M. bovis-BCG, and M. tuberculosishave been described and used for typing isolates.

The strategy used was to construct a vector which replicates as aplasmid in E. coli and as a phage in mycobacteria. One approach toaccomplishing this development of a shuttle plasmid was based on theidea that since mycobacterial DNA is not expressed well in E. coli, itshould be possible to clone, in a plasmid vector, a functionalmycobacteriophage genome which would not lyse the E. coli host. Itwould, thus, be able to replicate in both types of organisms. Becausetransfection of M. smegmatis would yield mycobacteriophage particles,introduction of DNA of interest into the slow growing mycobacteria(e.g., BCG) could be achieved by phage infection. A bifunctional vectorfor Streptomyces has been described by Suarez and Chater. Suarez, J. E.and K. F. Chater, Nature, 286: 527-529 (1980). A lambda-ColE1 vectorwith dual properties in E. coli has been referred to by Brenner andco-workers as a plasmid. Brenner, S. et al., Gene, 17: 27-44 (1982).

For this purpose, the mycobacteriophage TM4 was used. TM4 has beenreported to be a lysogenic phage isolated from M. avium. Timme, T. L.and P. J. Brennan, Journal of General Microbiology, 130: 205-209 (1984).It had been characterized as being a phage that lysogenizes M.smegmatis. It was shown to be capable of replicating in M. smegmatis,BCG, and M. tuberculosis and has been reported to be temperate. Thisphage also has a double stranded DNA genome of 50 kb and possessescohesive ends. It is possible, however, to use other mycobacteriophageshaving similar characteristics. The following procedures described asused with TM4 can also be used with such other mycobacteriophages inconstructing a vector.

The strategy used to introduce an E. coli plasmid replicon into phageTM4 to generate a vector that replicates in E. coli as a plasmid and inmycobacteria as a phage is schematized in FIG. 2. Plate stock lysatesand genomic DNA of TM4 phage were prepared as described for D29 phage(see Example 1). TM4 DNA was ligated at high concentrations to form longconcatamers of annealed cohesive ends. The ligated DNA was partiallydigested with Sau3A. Sau3A cuts the TM4 genome frequently (e.g., anaverage of once every 300 bp) to fragments 30-50 kb in size. Itgenerates a set of DNA fragments whose lengths were that of the entireTM4 genome or TM4 genomes with small deletions, but are cleaved at anyof the Sau3A sites within the genome. These DNA fragments were ligatedto the 6.5 kb cosmid pHC79, which contains the gene for resistance toampicillin and had been cleaved with BamHI. Hohn, B. and J. Collins,Gene, 9: 291-298 (1980). To select for recombinant molecules of theappropriate size, the ligation mixture was packaged into bacteriophagelambda heads in vitro. This selects for DNA fragments which containlambda COS sites and are between 38 and 53 kb in size. The resultingphage particles were transduced into E. coli and colonies containingpHC79::TM4 DNA molecules were selected on media containing ampicillin.Plasmid covalently closed circular DNA was isolated from 40,000 pooledampicillin-resistant (amp^(r)) colonies. Birnboim, H. and Doly, Journalof Nucleic Acid Research, 7: 1513-1525 (1979).

This library contains recombinant molecules of TM4 genomes into whichpHC79 cosmid DNA had been randomly inserted in Sau3A sites around theTM4 genome. It was transfected into M. smegmatis spheroplasts to selectfor TM4 phages which had pHC79 inserted in non-essential regions. Suchphages were, thus, shuttle phasmids. The transfection yielded 100 plaqueforming units (pfu) per ug of plasmid DNA. Plaque lifts were used toscreen for hybridization to ³² P-labelled pHC79.DNA; only 10 of 4000plaques hybridized to the labelled pHC79.

Following plaque purification and propagation on M. smegmatis cells, onesuch phage was studied in detail and designated as plasmid, phAE1.Plasmid phAE1 has been deposited (Feb. 26; 1986), according to the termsof the Budapest Treaty, at the American Type Culture Collection(Rockville, Md.) under accession number 40306. All restrictions onpublic access to the deposit will be removed irrevocably upon grant of aUnited States patent based on this application. DNA was isolated fromphAE1 phage particles grown on M. smegmatis, purified on CsCl gradients,ligated to form concatamers, and packaged in vitro into bacteriophagelambda heads. The resulting particles transferred ampicillin resistanceto E. coli cells and, when transfected, produced plaques on M.smegmatis. This was proof that phAE1 functions as a shuttle vector.

Restriction digests of phAE1 DNA isolated from phage particlespropagated on M. smegmatis and of phAE1 DNA isolated as plasmid DNAisolated from E. coli showed identical patterns, except for the presenceof unannealled fragments held together by the cohesive ends seen in thephage DNA preparation (FIG. 3A). Southern analysis demonstrated that thecosmid pHC79 was cloned within one of the two 11 kb KpnI restrictionfragments of the TM4 genome (FIG. 3B). By electron microscopy, the phAE1particles resemble bacteriophage lambda with hexagonal heads thataverage 50 um in diameter. However, these particles have long tails (180to 220 um in length) with a disc-like baseplate present on many of thetails (FIG. 4C). The structure is very similar to that of the parent TM4phage. Timme, T. L. and P. J. Brennan, Journal of Gen. Microbiology,130: 205-209 (1984).

Restriction analysis of DNAs from isolated phages resulting from thetransfection of the pHC79::TM4 library into M. smegmatis that did nothybridize to pHC79 showed them to be identical. The phage appears tohave resulted from a recombination event which occurred in transfectedcells containing two or more pHC79::TM4 molecules, yielding a wild-typeTM4 genome.

Of particular interest is the observation that the shuttle plasmid,phAE1, which was obtained from M. smegmatis, is like its parent TM4 inthat it is able to infect and replicate in three different M. bovis-BCGvaccine strains tested: the Glaxo, Pasteur, and Danish BCGs. Theseresults are presented in FIGS. 4A and 4B.

Thus, this demonstrates successful construction of E. coli-mycobacterialshuttle phasmids that are recombinant DNA molecules that not only havethe ability to replicate in E. coli as plasmids and in mycobacteria asphages, but also have the ability to be packaged into bacteriophagelambda heads or into mycobacteriophage particles. It also demonstratesthat recombinant DNA has been introduced into both a fast-growingmycobacterium (M. smegmatis) and a slow-growing mycobacterium (M.bovis-BCG). This makes it possible to infect BCG vaccine strains withthe shuttle phasmids and, thus, to introduce cloned genes intomycobacteria. Thus, this eliminates the need to develop a transfectionsystem for BCG. That is, because the E. coli-mycobacterial shuttleplasmid, upon transfection into mycobacteria is packaged intomycobacterial particles, DNA of interest can be introduced intoslow-growing mycobacteria (e.g., BCG) by transduction, rather thantransfection. Until now, this could not be done and this advance makesit possible to produce recombinant mycobacterial vaccine vehicles, whichcan be used to immunize against one or more antigens of interest.

The use of in vitro packaging to construct these phasmids can beextended as an efficient strategy for cloning of genes (e.g., genes, orDNA of interest, encoding an antigen or antigens for one or morepathogens against which an immune response is desired) into thesevectors, as long as the size limits of the packaging system are notexceeded. It is also possible, by screening additional TM4::pHC79recombinant phasmids, to determine the maximum amount of DNA that can bedeleted from the TM4 phage and to define additional non-essentialregions of the phage genome into which DNA can be inserted.

Introduction of new genes (e.g., DNA of interest encoding antigens) intomycobacteria by means of the shuttle plasmid entails cloning DNAfragments into the shuttle plasmid in E. coli and subsequentlytransfecting them into M. smegmatis spheroplasts. This yieldsrecombinant phage particles containing the cloned gene(s). Using theresulting M. smegmatis spheroplasts containing the recombinant phages,it is possible to infect BCG with high efficiency (approaching 100%efficiency), thus introducing DNA of interest included in therecombinant phages into BCG. Development of conditions for establishinglysogeny or recombination, to permit stable expression of the foreigngene(s) in mycobacterial cells, is highly desirable.

Introduction of DNA of interest into mycobacterial cells

The shuttle vectors described above and in the following sections can beused to introduce DNA of interest which encodes one or more antigens forone or more pathogens of interest into mycobacteria, such as M.bovis-BCG or M. smegmatis. It can also be used, by introducing DNAencoding appropriate antigens, such as human gonadotropin hormone (HGH)fragments, into mycobacteria, to produce an anti-fertility "vaccine."These vectors can also be used to introduce DNA encoding a protein or apolypeptide which is a growth inhibitor for or cytocidal to tumor cells.The resulting recombinant mycobacteria can be used, respectively, tonon-specifically augment immune responses to foreign antigens expressedin mycobacteria and to treat some human cancers. The shuttle vectorsprovide a means of manipulating and amplifying recombinant DNAconstructs in a bacterium (e.g., E. coli, Streptomyces, Bacillus), orother organism (e.g., yeast), and subsequently transfering them into amycobacterium where the DNA is expressed.

As a result, it is possible to produce recombinant mycobacterialvaccines which can be used to immunize individuals against, for example,leprosy, tuberculosis, malaria, diphtheria, tetanus, leishmania,salmonella, schistomiasis, measles, mumps, herpes, and influenza. Genesencoding one or more protective antigens for one or more of thedisease-causing pathogens can be introduced into the mycobacterium. Ofparticular value is the ability to introduce genes encoding antigens ofpathogens which require T-cell memory or effector function.Administration of the resulting recombinant mycobacterial vaccine to ahost results in stimulation of the host's immune system to produce aprotective immune response.

A vaccine against a pathogen or toxin can be produced, using the shuttleplasmid of the present invention, by the following procedure: DNAencoding an antigen (or antigens) for the pathogen or toxin againstwhich protection is desired is obtained. The DNA can be obtained byisolation of the naturally-occurring DNA (e.g., from the pathogenicorganism or toxin-producing organism); by cloning and amplification ofthe DNA sequence of interest, using known genetic engineering techniques(See, for example, Maniatis, T. et. al. Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y. (1982).); or by mechanical synthesis.

By the following procedure, the gene or genes of interest (i.e., whichencode one or more antigens against which immunity is desired) arecloned into the shuttle plasmid. This can be explained with reference toFIG. 2, which is a schematic representation of the shuttle plasmidphAE1. The cohesive ends of the shuttle plasmid are ligated, using knowntechniques. The resulting shuttle plasmid material is cut (digested)with a unique restriction enzyme (e.g., a restriction enzyme which cutsat unique sites, such as EcoRI and EcoRV, in the shuttle plasmid). Analternative approach to cuts made in this way is the addition (byligation) of a polylinker (oligonucleotide sequence) which is usefulwith (can be cut by) other restriction enzymes. In this case, the linkeris cut open with a selected restriction enzyme, producing sites at whichDNA of interest can be inserted.

In the first case (cutting using a unique restriction enzyme), theresult is shuttle plasmid molecules which have been cut once and intowhich DNA of interest can be inserted. In the second case, there is alsoat least one site at which DNA can be inserted. Anantibiotic-resistance-encoding gene (e.g., anampicillin-resistance-encoding gene) and DNA encoding one or moreantigens, against which immunity is desired, can be ligated, using knowntechniques, at the restriction sites. The DNA being inserted and theshuttle plasmid DNA are generally ligated in equal molar amounts.

The resulting ligated DNA, which in this case includes the shuttleplasmid DNA, an antibiotic resistance gene and antigen-encoding DNA, ispackaged into bacteriophage lambda heads using lambda in vitro packagingmix. E. coli is subsequently transduced with the phage, with the resultthat it is possible to screen (using antibiotic-containing medium) forcolonies containing the antibiotic-resistance-encoding gene and theantigen-encoding DNA.

The resulting "library" is introduced into M. smegmatis using, forexample, electroporation. Plaques which contain shuttle phasmidscontaining cloned insert DNA are selected. Subsequently, recombinant M.smegmatis can be used to infect a cultivable mycobacterium, such as BCG,with high efficiency. As a result, the antigen-encoding DNA isintroduced into mycobacterial genomic DNA, where it will be expressed.

Selection of BCG containing the DNA of interest (here DNA encoding oneor more antigens integrated into their genomic DNA) can be carried outusing a selectable marker. One approach to selection of BCG containingDNA encoding one or more antigens, introduced by infection with therecombinant phage, is based on use of a selectable marker, which is anantibiotic resistance gene. In this case, the shuttle plasmid includes agene encoding, for example, kanamycin resistance, viomycin resistance,thiostrepton resistance, hygromycin resistance, or bleomycin resistance.

A second approach in which a selectable marker is used to select BGGcontaining the DNA of interest is an auxotrophy strategy (i.e., onewhich relies on use of a mutant microorganism which requires somenutrient or substance not required by the organism from which the mutantwas derived). In this case, a mycobacterium having the mutation is usedand the gene which encodes the missing or mutated function isincorporated into the shuttle plasmid (which also containsantigen-encoding DNA). Selection for mycobacteria containing theantigen-encoding DNA is thus based on the ability of mycobacteria intowhich the shuttle plasmid is successfully introduced to survive, whengrown on appropriate media.

For example, a system which includes a host mutant (e.g., M. smegmatis,BCG) and a selectable marker that complements the mutation can be used.Such a system can include a host mutant which is a pyrF⁻ BCG mutant anda selectable marker, such as a pyrF⁺ gene, present in the plasmidshuttle vector used to introduce the antigen-encoding DNA into the(mutant) BCG. For example, the plasmid can include, in addition to theantigen-encoding DNA inserted into cosmid DNA, the pyrF⁺ gene. Thus, BCGmutants into which the plasmid is introduced by infection can beselected by plating on minimal media. An alternative approach is to use2-deoxyglucose-resistant mutants; in this case, the mycobacterialglucokinase gene is cloned into the plasmid and is used for selection,as described above for pyrF.

Selection on this basis will result in BCG having the antigen-encodingDNA stably integrated into genomic DNA and expressed by the bacillus.For this, gene expression signals (e.g., promoters, ribosome bindingsites) are included upstream of the foreign (antigen-encoding) DNA, toenable BCG containing the antigen-encoding DNA to express it at levelssufficient to induce an immune response in a host to whom it isadministered.

It is also possible to select BCG containing DNA encoding one or moreantigens by use of monoclonal antibodies. In this case, a gene or genefragment encoding one or more epitopes of an antigen (e.g., M. leprae orM. tuberculosis) for which monoclonal antibodies are available isintroduced into the mycobacteria. Such monoclonal antibodies are used toselect for recombinant BCG containing a gene or genes encoding one ormore of these epitopes. The antigen genes introduced in this way containa promoter sequence and other regulatory sequences. As a result,additional series (e.g., DNA encoding other antigens) can be added,using genetic engineering techniques, in frame, such that recombinantBCG identified by monoclonal antibodies to one antigen would also beexpressing other foreign antigen-encoding DNA so introduced.

A parallel strategy which makes use of a plasmid to introduceantigen-encoding DNA into cultivable mycobacteria can also be used tomake a vaccine vehicle. This will result in stable maintenance of theDNA of interest extrachromosomally as a plasmid and its subsequentexpression.

Construction of such a shuttle plasmid is represented schematically inFIG. 8. In this case, a selectable marker, which would make it possibleto select cells containing the antigen-encoding DNA, is used. Theselectable marker can be, for example, an antibiotic-resistance-encodinggene or a gene which complements that missing in an auxotrophic mutant,as described above with reference to the shuttle plasmid. In theauxotrophy strategy, an auxotrophic mycobacterial mutant (e.g., a pyr⁻ Fmutant) is isolated and the gene present in the corresponding wild-type(nonmutant) mycobacterium is incorporated into the plasmid. In additionto the pyr⁻ F mutant, it is possible to isolate deoxyglucose mutants,which have a defect in the glucokinase gene, as well as others havingmutations in other biosynthetic pathways (e.g., mutations in amino acidbiosynthesis, vitamin biosynthesis and carbohydrate metabolism, such asarabinose and galactose).

In either approach, a mycobacterial mutant is selected and the genewhich complements the mutation is incorporated into the plasmid vector,which also contains the antigen-encoding DNA of interest. Themycobacterial mutants into which the antigen-encoding DNA issuccessfully introduced will be identifiable (can be selected) byculturing on appropriately-selected media (e.g., media containing theantibiotic against which resistance is conferred, media containing orlacking the nutrients involved in the biosynthetic pathway affected inthe mutant used) or by selecting on the basis of the appearance ofplaques formed, when the cI gene is used.

Another component of a plasmid useful in introducing antigen-encodingDNA into the recombinant mycobacteria vaccine vehicle is an autonomouslyreplicating sequence (e.g., a replicon), whose presence is a keydeterminant in allowing the plasmid to replicate autonomously(extra-chromosomally). These sequences can include, for example, aplasmid replicon, segments of a mycobacteriophage or chromosomalreplication origins.

The design of the shuttle plasmid phAE1 includes several of thesefactors. For example, introduction of the E. coli cosmid pHC79 into themycobacteriophage TM4 made it possible to provide an E. coli plasmidreplicon origin and a selectable ampicillin resistance gene, as well asthe bacteriophage lambda cohesive (cos) sequences and a unique EcoRIsite. There are no EcoRI sites within the TM4 phage; the unique EcoRIsite within phAE1 can be used for introducing foreign gene(s) into theplasmid. As described in Example 4, a 1.6 kb EcoRI fragment encoding theaminoglycoside phosphotransferase (aph) gene from Tn903 has been clonedinto phAE1 using this cosmid cloning strategy.

There are several useful approaches to efficiently introduce theantigen-encoding DNA into a cutivable mycobacterium, such as M.bovis-BCG or M. smegmatis, which is to be used as a vaccine vehicle. Forthe plasmid, which includes DNA encoding the antigen(s) of interest, aselectable marker and an autonomously replicating sequence, protoplastfusion can be used for efficient introduction into the mycobacterium. Inthis case, E. coli or streptomyces having a cloned plasmid is fused,using known techniques, with a mycobacterial spheroplast. Using thisapproach, it is possible to transfer the foreign (antigen-encoding) DNAinto the mycobacterium. Alternatively, E. coli minicells, which containplasmid DNA and essentially no chromosomal DNA, can be used in carryingout a minicell protoplast fusion.

If, in the alternative, DNA of interest can be moved efficiently intothe mycobacterium, an autonomously replicating sequence is not necessaryand, instead, the DNA of interest (e.g., antigen-encoding) can beintegrated into the mycobacterial chromosomes. This can be accomplished,for example, using minicell protoplast fusion. In this case, aselectable marker for the mycobacterium, which can be anantibiotic-resistance gene or a chromosomal mutation, can be cloned intoan E. coli cosmid. Also present in the E. coli cosmid will be DNA whichallows efficient integration of DNA of interest into the mycobacterialchromosome. For example, in M. leprae, a repetitive sequence occurswhich appears to be associated with recombination; analogous sequencescan be identified in and isolated from BCG and M. smegmatis,incorporated into the E. coli cosmid (along with the selectable marker)and result in a high degree of recombination.

A gene or genes of interest (encoding one or more antigens) can beincorporated into the construct described (e.g., which includes an E.coli replicon, a segment of mycobacterial chromosomal DNA associatedwith recombination (a recombinogenic sequence) and two selectablemarkers-one serving as a marker in E. coli and the second serving as amarker in the mycobacterium). The gene(s) can then be integrated intomycobacterial chromosomal DNA, such as BCG or M. smegmatis chromosomalDNA. If the gene(s) of interest are integrated in this way into M.smegmatis, it/they can also be moved into BCG by means of a generaltransducing phage. In this case, it is preferable to include, inaddition to the other construct components, two recombinogenicsequences: one from M. smegmatis and one from BCG.

Methods of producing and using the recombinant mycobacteria of thepresent invention are described in detail in the following sections.Those sections describe and exemplify: introduction of DNA of interestinto mycrobacteria by means of a shuttle plasmid, to integrate the DNAof interest into mycobacterial chromosomal DNA; introduction of DNA ofinterest into mycobacteria by means of a shuttle plasmid, to producemycobacteria in which the DNA of interest is expressed episomally;introduction of DNA of interest into mycobacteria by means of arecombinant plasmid vector, to integrate DNA of interest intomycobacterial chromosomal DNA through homologous recombination;characteristics of the recombinant mycobacteria; and uses of suchproducts.

Introduction of DNA of interest by means of a shuttle plasmid

The present work has resulted in identification of a mycobacteriophagewhich stably lysogenizes M. smegmatis. One such phage, L1, hadpreviously been reported to produce turbid plaques on M. smegmatis andputative lysogens were resistant to superinfection and could be inducedto produce phage. Doke, S. J. Kunamoto Med. Soc. 34, 1360-1373 (960).Tokunaga, T. & Sellers, M. I. in Host-Virus Relationships inMycobacterium, Norcardia, and Actinomycetes (eds. Juhasz, S. E. &Plummer, G.) 227-243 (Charles C. Thomas, Springfield, Ill., 1970). Theseobservations have been confirmed and, in addition, Southern analysis hasdemonstrated that a prophage is integrated in the M. smegmatischromosome (FIG. 5, Panel B, lanes 2, 3). Analysis of multipleindependent lysogens revealed identical patterns of unique bandsresulting from the phage integration, which suggests that L1 phageintegration is site-specific (FIG. 5, Panel B).

L1 has, thus, been shown to stably lysogenize M. smegmatis. L1-shuttlephasmids were constructed by introducing the E. coli cosmid, pHC79,containing a ColE1 origin of replication and an ampicillin-resistancegene for selection in E. coli, into a non-essential region of theL1-genome (FIG. 6). The L1-shuttle plasmid, designated phAE15, was shownto replicate in E. coli as a plasmid and in mycobacteria as a phage and,like the parent phage, integrates into the M. smegmatis chromosome (FIG.5, Panel B, lanes 4, 5). L1 phage is devoid of EcoRI sites. Theintroduction of pHC79 provided a unique EcoRI site for the pHAE15shuttle vector, making it possible to introduce genes of interest. Ithas been shown that these genes can be introduced and stably maintainedwithin the mycobacteria upon lysogenization with the shuttle plasmidvector (FIG. 6).

A 1.6 kb fragment containing the aminoglycoside phosphotransferase (aph)gene from Tn903, which confers kanamycin-resistance in E. coli, wasintroduced into phAE15 by a cosmid cloning strategy. Oka, A., Sugisaki,H. & Takanami, M. J. Mol. Biol. 147, 217-226 (1981) The aph gene withEcoRI ends was ligated to linear phAE15 DNA and packaged in vitro intobacteriophage lambda-heads. The resulting recombinant molecules weretransduced into E. coli. Closed circular plasmid DNA was isolated froman E. coli clone that was resistant to both ampicillin and kanamycin,and was transfected into M. smegmatis protoplasts. This resulted inmycobacterial phage particles which had packaged plasmid DNA. Thisplasmid; designated phAE19, has the ability to lysogenize M. smegmatiscells and generate kanamycin-resistance colonies (FIG. 7).Mycobacteriophages induced from these lysogens co-transduced immunity toL1 infection and kanamycin-resistance to sensitive M. smegmatis cells,demonstrating that the resistance to kanamycin results from expressionof the cloned aph gene. phAE19 can also lysogenize and conferkanamycin-resistance upon M. bovis-BCG. Kanamycin-resistance thusrepresents the first useful selectable marker for the mycobacteria. Inaddition, these results demonstrate that lysogeny is one means by whichDNA of interest can be introduced and expressed in mycobacteria.

Introduction of DNA of interest by means of a shuttle plasmid

Introduction of DNA of interest by means of a shuttle plasmid extendsthe capabilities of phages by offering increased cloning capacity, easeof DNA manipulation, and increased copy number. Plasmids from M.smegmatis have not been described previously and genetic manipulation inmycobacteria is difficult. Therefore, a shuttle plasmid vector capableof replicating and expressing DNA of interest in both E. coli andmycobacteria was constructed as follows. In order to ensure a functionalreplicon for mycobacteria, an E. coli plasmid, pIJ666, containing theneomycin/kanamycin phosphotransferase II (neo) gene from Tn5, and theP15A origin of replication and the chloramphenicol acetyltransferase(cat) gene from pACYC184, was inserted randomly into the plasmidpAL5000, which replicates in M. fortuitum. Kieser, T. and R. E. Melton,Gene, 65:83-91 (1988); Berg, D. E. et al., Proc. Natl. Acad. Sci.U.S.A., 72:3628-3632 (1975); Chang, A. C. Y. and S. N. Cohen, J. Bact.,134:1141-1156 (1978); Labidi, A. et al., FEMS Microbiology Letters,30:221-225 (1985). FIG. 8 outlines the construction of thepIJ666::pAL5000 library.

Transformation of this library into M. smegmatis spheroplasts has beendifficult, possibly due to the problem of regenerating viable cells. DNAwas therefore introduced directly into intact M. smegmatis cells byelectroporation to obviate possible damage to mycobacterial cells whichmight result from use of protocols for producing spheroplasts.Conditions were developed for electroporation of lytic phage DNA thatyielded more than 5×10³ pfu/ug. Electroporation of the pIJ666::pAL5000library under these conditions into M. smegmatis yielded kanamycin- andchloramphenicol-resistance transformants. Plasmid DNA isolated frompools of M. smegmatis transformants in three separate experiments wastransformed back into E. coli, selecting for kanamycin-resistance.Although pIJ666 was inserted at different sites within the pAL5000genome in many of the isolated E. coli transformants, all plasmids werestable in both species (FIG. 9). These methods have made it possible totransform some BCG vaccine strains with the pIJ666::pAL5000 recombinantlibrary, with expression of kanamycin-resistance, as described inExample 9 and shown in FIG. 10. Panel A of FIG. 10 showskanamycin-resistant BCG colonies which arose after electroporation ofBCG cells with shuttle plasmid DNA; panel B shows kanamycin-resistantBCG colonies that arose after electroporation without shuttle plasmidDNA. Using a similar approach, the 65 kD M. leprae gene has beenintroduced into BCG, in which it was expressed, as shown by resultspresented in FIG. 17.

Plasmid vector for integration of DNA of interest into mycobacterialgenomic DNA

A plasmid vector, which has been used to integrate DNA ofnonmycobacterial origin (i.e., from a source other than the mycobacteriainto which it was integrated) in mycobacterial genomic DNA, wasconstructed as represented in FIG. 11. Isolation of the M. bovis-BCGPyrF gene was carried out as follows and as described in Example 10. M.bovis-BCG DNA was partially digested with a restriction enzyme Sau3A,size selected-and inserted into the vector pUC19. The resulting librarywas used to transform E. coli cells which had an insertion in the E.coli PyrF gene. Four independent colonies which had acquired the abilityto grow in the absence of uracil were identified and plasmid DNA wasisolated from them. This plasmid DNA was used to construct therecombinant plasmid vector. The PyrF gene of M. smegmatis wasincorporated into the pUC19 plasmid vector at the BamHI site and thekanamycin resistance gene (Kan) was inserted into the PyrF gene at theBamHI site, using known techniques. PyrF⁺ cells are able to grow inmedium without uracil and are fluoro-orotic acid sensitive (FOA^(S));PyrF⁻ cells need uracil for growth and are fluoro-orotic acid resistant(FOA^(R)). Cells containing the kanamycin resistance gene are kamanycinresistant (KAN^(R)) and those without the gene are kanamycin sensitive(KAN^(S)). Ausubel, F. M. et al. (ed.) Current Protocols in MolecularBiology, p. 1.5.4, Green Pub. (1987). Plasmid DNA containing DNA frompUC19, Mycobacterium smegmatis and Tn903, designated pRH1100, has beendeposited, according to the terms of the Budapest Treaty, at theAmerican Type Culture Collection (Rockville, Md.) under accession number40468 (deposit date Jul. 6, 1988). All restrictions on public access tothe deposit will be removed irrevocably upon grant of a United Statespatent based on this application.

Stable Integration of DNA of Nonmycobacterial Origin into MycobacterialGenomic Material

As described below and in Example 10, electroporation was used tointroduce the resulting recombinant plasmid vector into mycobacteria. Asrepresented in FIG. 12A, in cells transformed with the recombinantplasmid, homologous recombination occurred between sequences on theincoming recombinant plasmid containing the PyrF gene and homologousmycobacterial chromosomal (genomic) sequences, in integration of theincoming PyrF and Kan sequences. Mycobacterial cells containing theintegrated recombinant plasmid, which contains the Kan gene, wereselected by culturing the electroporated cells on kamanycin-containingmedium. Only those cells in which integration of the Kan gene occurredsurvived.

Mycobacterial cells in which the DNA of interest (here, the Kan gene)were identified as follows. The entire integrated recombinant plasmid isunstable because the mycobacterial genome into which it integratescontains two identical sequences in close proximity to one another. As aresult, recombination of homologous sequences can again occur. Thisresults in looping out (also called resolution), which results inremoval of the recombinant plasmid, producing no net change in themycobacterial genome, or in removal of the recombinant plasmid in such amanner that the Kan-containing PyrF gene remains in the mycobacterialgenome. Resolution occurs with low frequency, but cells in which it hasoccurred can be identified and isolated on the basis of the phenotypethey exhibit. PyrF⁺ cells (those in which no net change in the genomeresults), as indicated in FIG. 12, will be kanamycin sensitive andfluoro-orotic acid sensitive (FOA^(S)). Cells in which resolutionresults in integration of the Kan-containing PyrF gene are kanamycinresistant and exhibit FOA^(R) because the PyrF gene is disrupted and,thus, nonfunctional. Thus, plating of the KAN^(R) mycobacterialpopulation on FOA-containing medium will result in identification ofcells in which the Kan gene is stably integrated into genomic DNA (FIG.12B, lower left: KAN^(R), FOA^(R)).

Thus, the Kan gene was stably integrated into the mycobacterial genome,using homologous recombination of adjacent PyrF sequences. An importantadvantage of the method of the present invention, which is illustratedin FIG. 12B, is that integration of the DNA of interest occurs withoutconcomitant integration of plasmid or phage DNA into the genome. Thatis, the net effect is that the plasmid sequences are not present in therecombinant mycobacterial cells. Expression of the Kan gene was alsodemonstrated and cells in which both integration and resolution hadoccurred selected for on the basis of cell phenotype (in this case,KAN^(R), FOA^(R)). In the work described above, DNA of nonmycobacterialorigin (i.e., kanamycin resistance gene) was successfully introduced andstably integrated into M. smegmatis genomic DNA. The same techniques canbe used to introduce DNA of nonmycobacterial origin into M. bovis-BCG orother mycobacterial genomic DNA.

Stable Integration of DNA Encoding an Antigen or Antigens intoMycobacterial Genomic DNA

Integration of an Interrupted PyrF Gene

In a similar manner, DNA encoding one or more antigens against which animmune response is desired can be integrated into mycobacterial genomicDNA. The method of the present invention, by which DNA of interest isintegrated into mycobacterial genomic DNA, is represented schematicallyin FIG. 13. The method will be described with particular reference tointegration of DNA which is the 65 KD M. leprae gene into M. smegmatis,which has been carried out (see Example 11). It is to be understood,however, that the same approach can be used to introduce the M. leprae65 KD gene into other mycobacteria, as well as to integrate DNA encodingother polypeptides or proteins against which an immune response issought into M. bovis-BCG, M. smegmatis or other mycobacteria.

Integration of DNA encoding a selected antigen (designated Fan, forforeign antigen) is represented in FIG. 13. An appropriate plasmidvector (e.g., one which can replicate in E. coli but not inmycobacteria), such as the recombinant pUC19 plasmid represented in FIG.13, is used. The recombinant plasmid includes a mycobacterial gene orDNA sequences such as the PyrF gene represented in FIG. 13; sequences inthis gene, which are homologous to those in the mycobacterial genome,provide the basis for homologous recombination between plasmid-bornemycobacterial sequences and genomic mycobacterial sequences to occur.The recombinant plasmid also includes DNA sequences necessary forreplication and selection in E. coli and DNA sequences necessary forselection in mycobacteria. The sequences for use in selection confer adistinctive phenotype on the cell, thus making it possible to identifyand isolate cells containing the gene. The gene can encode, for example,drug resistance. In FIG. 13, the recombinant plasmid includes a geneconferring kanamycin resistance, thus making it possible to selectmycobacteria containing the gene simply by culturing onkanamycin-containing medium. The recombinant plasmid also contains DNAencoding one or more polypeptide or protein against which an immuneresponse is desired (designated Fan), which is integrated intomycobacterial genomic DNA.

In one embodiment of the present invention, the 65 KD gene of M. lepraehas been integrated into M. smegmatis genomic DNA through use of arecombinant plasmid as represented in FIG. 13, in which Fan is the M.leprae gene.

The recombinant plasmid (e.g., a plasmid containing the PyrF gene intowhich the 65 KD M. leprae gene and a Kan gene were inserted) wasintroduced into mycobacterial cells (M. smegmatis) using standardelectroporation techniques. (See Example 11). Electroporated cells werethen plated onto kanamycin-containing medium. Only kanamycin-resistant(KAN^(R)) cells grew under these conditions; such cells had integratedinto genomic DNA the KAN^(R) gene and the M. leprae gene and were alsoFOA^(S) (due to the disrupted PyrF genes from the recombinant plasmidand from the mycobacterium).

Cells were subsequently transferred to FOA-containing medium to identifythose cells in which the Fan gene (here, the M. leprae gene) was stablyintegrated into genomic DNA. As indicated in the bottom panel of FIG. 13(left side), integrated into genomic DNA of such cells (KAN^(R),FOA^(R)) is the disrupted PyrF gene which contains the kanamycinresistance gene and the Fan gene. As indicated at the right side of thebottom panel, mycobacterial cells which have undergone looping out withthe result that only a complete PyrF gene remains in the genome are bothkanamycin sensitive and fluoro-orotic acid sensitive.

Thus, as described above and in Example 11, it has been possible tointegrate into mycobacterial genomic DNA DNA encoding a protein antigenand to identify and select those cells which contain the stablyintegrated DNA of interest. In addition, such DNA of interest has beenintegrated into the mycobacterial genome at a selected site (in thiscase, at the PyrF gene site). This same approach can, of course, be usedto integrate DNA of interest into other selected sites on mycobacterialgenomic DNA. In this case, a site on the genome, at which integration isdesired, can be selected. A recombinant plasmid containing sequenceshomologous to or sufficiently similar to the selected genomic sequences;DNA of interest; sequences necessary for replication and selection in E.coli; and DNA sequences necessary for selection in mycobacteria can beconstructed, as described previously. The DNA of interest can be stablyintegrated into mycobacterial genomic DNA and cells containing thestably integrated DNA of interest selected, in the same manner asdescribed previously.

It is possible to introduce all or a portion of any gene whoseexpression in mycobacteria is sought into mycobacteria using the samemethod. That is, the following method can be used to stably integrateinto a mycobacterial genome DNA of interest:

A recombinant plasmid vector, which can replicate in E. coli but not inmycobacteria and which includes:

1. a mycobacterial gene, or portions thereof, necessary forrecombination with homologous sequences in the genome of mycobacteriatransformed with the recombinant plasmid;

2. all or a portion of a gene which encodes a polypeptide or proteinwhose expression is desired in mycobacteria transformed with therecombinant plasmid;

3. DNA sequences necessary for replication and selection in E. coli; and

4. DNA sequences necessary for selection in mycobacteria (e.g., drugresistance).

is used to transform mycobacterial cells, such as M. smegmatis or M.bovis-BCG. The recombinant plasmid is introduced into mycobacterialcells using known techniques. In one embodiment, the plasmid isintroduced by means of electroporation, using standard bacterialelectroporation procedures. (See Example 11).

Electroporated cells are plated under conditions which allow selectionof cells in which integration has occurred. As described above, theplasmid can contain a gene encoding drug resistance; such as kanamycinresistance. In that instance, electroporated cells are plated ontomedium containing kanamycin. Only kanamycin resistant (KAN^(R)) cells,which are cells in which plasmid DNA has been integrated, will surviveunder these conditions.

Surviving cells are subsequently plated under conditions which make itpossible to identify and select those in which the DNA of interest isstably integrated in genomic DNA. In the case in which the PyrF gene hasbeen disrupted by insertion of DNA of interest, surviving cells areplated onto FOA-containing medium, which makes it possible to identifycells in which resolution has occurred because they are FOA^(R) (and,thus, grow in such medium).

The DNA encoding a polypeptide or protein against which an immuneresponse is sought, which is present in the recombinant plasmid, can beisolated from a source in which it occurs in nature, produced by meansof standard genetic engineering techniques, in an appropriate host, orsynthesized chemically or mechanically. Similarly, plasmid-borne DNAsequences necessary for homologous recombination can be isolated from asource in which it occurs in nature, produced by means of standardgenetic engineering techniques or synthesized chemically ormechanically. The characteristic which serves as the basis for selectionof mycobacterial cells containing integrated DNA of interest can be, asdescribed, drug resistance. The gene can encode, for example, kanamycinresistance, viomycin resistance, thiostrepton resistance, hygromycinresistance or bleomycin resistance. Alternatively, an auxotrophystrategy can be used, such that selection is based on the ability ofmycobacteria in which integration has occurred to survive, when grown onappropriate medium.

Integration of a PyrF-DNA of Interest (Fan) Combination

An alternative approach to that described above, in which amycobacterial gene (e.g., PyrF) is disrupted by a drug resistance geneand DNA of interest, is one in which DNA of interest is integrated intoa mycobacterial genome without additional sequences (e.g., without theKan gene), as occurs as a result of the earlier described method. Thismethod is represented in FIG. 14.

In this method, recombinant mycobacterial cells which are targets forfurther manipulation and introduction of DNA of interest are firstproduced. This can be done, for example, by making a precise replacementof the mycobacterial PyrF gene by a kanamycin resistance gene. Standardrecombinant DNA techniques are used in this replacement procedure, inwhich sequences flanking the PyrF gene are used to insert the Kan gene.The recombinant plasmid (in which the PyrF gene is replaced with Kan) isintroduced into mycobacterial cells using standard electroporationmethods. The resulting electroporated cells are plated onto mediumcontaining kanamycin and no uracil. Mycobacterial cells in which boththe kanamycin resistance gene and the genomic PyrF gene are present willbe selected at this point. All other cells (KAN^(R) Ura⁻ ; KAN^(S) Ura⁻; KAN^(S) Ura⁺) will die. Cells selected in this manner are subsequentlyplated onto medium containing kanamycin, fluoro-orotic acid and uracil.As shown in FIG. 14, this results in selection of mycobacterial cellswhich are URA⁻ and FOA^(R) (because they contain no PyrF gene), as wellas KAN^(R) (because of the integrated Kan gene).

Mycobacterial cells produced in this manner are used in this method astargets (target mycobacterial cells) for further manipulation, usingknown techniques, by which DNA of interest, and an intact PyrF gene areintegrated into mycobacterial genomic DNA. As represented in FIG. 15, arecombinant plasmid, similar to that described previously and in Example10, which includes an intact PyrF gene and DNA of interest (next to orclosely following one end of the PyrF gene) is used. The recombinantplasmid is introduced into the "target" mycobacterial cells (whichinclude a Kan gene and no PyrF gene) using standard techniques (e.g.,electroporation). Homologous recombination occurs between sequences toone side of the Kan gene present in the target mycobacterial cells andto one side of the PyrF gene present in the recombinant plasmid,resulting in integration into the target mycobacterial cells genomes ofthe PyrF gene-DNA of interest combinations, as represented in FIG. 15.

Electroporated cells are plated onto medium containing kanamycin and nouracil. Only those cells which contain the kan gene and the PyrF gene(with which the DNA of interest has entered the cells) will surviveunder these conditions. Subsequent culturing of survivors on mediumcontaining no added uracil will result in growth of only thosemycobacteria having integrated into their genomes the PyrF gene-DNA ofinterest combination, as represented in FIG. 15.

In those cases in which DNA of interest is from a source which resultsin its inability to be expressed in mycobacteria, an expression cassettecan be used. The expression cassette can contain a mycobacterialpromoter and ribosome binding site, which will serve as expressionsignals controlling expression of the DNA of interest. As represented inFIG. 16, the expression cassette can include a polylinker in sequencessurrounding the pyrF gene. As a result, DNA of interest can be insertedand the mycobacterial signals will control its expression. Selection ofmycobacterial cells in which the PyrF-expression cassette-DNA ofinterest combination are stably integrated can be carried out asdescribed previously in relation to FIG. 15.

A slightly different, but related method by which DNA of interest can beintegrated into mycobacterial genomes makes use of mycobacteria fromwhich the normally present PyrF coding sequences normally present havebeen removed, using known techniques. A recombinant plasmid similar tothat described previously except that it includes an intact(undisrupted) PyrF gene in combination with DNA of interest (located ator near one end of PyrF), is introduced into PyrF-deleted mycobacteria(e.g., by electroporation). Cells which contain an intact PyrF gene(and, thus, DNA of interest) are identified by culturing electroporatedcells on medium containing no uracil. Only cells containing the PyrFgene will survive. Subsequent growth on medium containing no uracil willalso identify those cells in which looping out has resulted in stableintegration of PyrF and the DNA of interest into the mycobacterialgenome.

The outcome of both of these latter two approaches, in which PyrF isreplaced with a Kan gene in or PyrF is deleted from the mycobacterialgenome is that the resulting recombinant mycobacterial genome includes afunctional PyrF gene and the DNA of interest, but does not contain agene encoding drug resistance (e.g., Kan) or other selectable marker.

Overview of uses and advantages of shuttle vectors and methods of thepresent invention

There are numerous uses for and advantages of the plasmid and theplasmid vectors of the present invention, as well as for the method ofthe present invention in which they are used. These are described belowand their use in constructing vaccine vehicles is described in thefollowing sections. As a result of the present invention, by which DNAintroduced into mycobacteria has been expressed, new genetic approachesto understanding questions of disease pathogenesis are now available.Using either phage or plasmid vector systems, it should be possible toinsertionally mutagenize and mark genes of pathogenic mycobacteria,either by homologous recombination or by transposon mediated insertionor deletion, with the aim of identifying specific genetic functionsrequired for virulence and pathogenesis. For example, using thesevectors and mycobacteria (e.g., M. smegmatis, M. bovis-BCG), virulencegenes of M. tuberculosis or M. leprae can be identified and diagnostics(diagnostic tests) developed. By specifically deleting or replacingthose genes, it may be possible to develop a more specific and effectiveattenuated vaccine against tuberculosis than the current M. bovis-BCGvaccine. Alternatively, as specific protective antigens for tuberculosisand leprosy are identified by study of antigens recognized by T cellsfrom resistant individuals, it will now be possible to introduce andexpress them in currently existing M. bovis-BCG vaccines.

The vectors of the present invention are the first to make it possibleto construct genomic libraries in a mycobacterium. This is particularlyuseful in identifying antigens or enzymes or drug targets for apathogenic mycobacterium. For example, in the case of M. leprae, genomicDNA is sheared and cloned, to ensure that the entire genomic DNA isincluded. Using the subject vectors, the library of the M. lepraefragments is first introduced into a bacterial host, such as E. coli,where it is expressed. It is subsequently moved into a mycobacterium(e.g., M. smegmatis, BCG). As a result, the library exists in amycobacterial host, thus making it more efficient to look formycobacterial antigens, enzymes, drug targets and diagnostic probes.

A shotgun approach can be used to introduce DNA into BCG and to identifyclones containing genes which enable them to grow faster thanpresently-available BCG, which is a slow-growing mycobacterium. Genesidentified in this manner can subsequently be used to produce BCG whichgrows faster than presently-used BCG. A similar approach can be used toclone M. leprae genes into a cultivable mycobacterium and to identifythose recombinant cells which grow. This approach can be used to make M.leprae from a cultivable mycobacterium, thus alleviating the presentdifficulty of producing the pathogen.

The vectors of the present invention can also be used to identify newdrugs for the prevention or treatment of tuberculosis or leprosy. Forexample, it is possible that a target against which a drug should bedirected is an enzyme (e.g., gyrase) produced by the causativemycobacterium. The corresponding enzyme-encoding genes in M. smegmatiscan be replaced, using the subject vectors, with the M. tuberculosis orthe M. leprae enzyme-encoding gene(s). This results in production of arecombinant M. smegmatis which can be used for testing to identify drugseffective against the enzymes (as well as for drugs effective against M.avium and M. intracellulare).

The genetic approaches described herein have made it clear that both theaph and neo genes encoding kanamycin resistance can be (and have been)stably expressed in M. bovis-BCG vaccine strains.

As a result of the work described herein, selectable markers (genesencoding identifiable characteristics on which selection can be based)are now available for mycobacteria. Three such selectable markers arenow available for mycobacteria: 1) the chloramphenicol acetyltransferase or cat gene, which confers chloramphenicol resistance; 2)the aminoglycoside phosphotransferase or aph gene from Tn903, whichconfers kanamycin resistance; and 3) the cI gene, which encodes therepressor protein of the L1 bacteriophage. Growth on medium containingthe appropriate drug is used in the cases of the drug resistance genesto select mycobacteria containing the cat or the aph gene. Selection ofvariants on the basis of the appearance (cloudy or clear) of the plaquesthey form when cultured is used in the case in which the cI gene is usedfor selection.

Also as a result of the work described herein, lysogeny and sitespecific recombination have been shown to occur between specific sites,known as attachment, or att sites, on the mycobacterial chromosomal DNAand on the mycobacterial phage. In bacteria infected with phage lambda,the physical condition of the phage DNA is different in the lytic andlysogenic states. Change from one of these states to the other involvessite-specific recombination. Integration of lambda DNA into host DNA(resulting in the free lambda DNA's becoming prophage DNA) must occurfor lysogeny to occur; conversely, excision of prophage DNA from thehost chromosome must occur for lysis to occur. Integration and excisioninvolve site-specific recombination. Although this phenomenon is knownfor several genera of bacteria, this is the first time it has beendemonstrated for mycobacteria. It can be used as a means by which DNA ofinterest can be incorporated into mycobacteria efficiently and stably.

For example, this can be accomplished by use of what is referred to as acos-att vector. Such a vector can include: 1) an E. coli or otherappropriate bacterial origin of replication; 2) a gene encoding aselectable marker, such as ampicillin resistance, for selection in E.coli or other bacterial host; 3) the att region of a temperate phage,such as L1; 4) the lambda cos site, in order that lambda in vitropackaging systems can be used; and 5) a gene encoding a selectablemarker, such as kanamycin resistance, chloramphenicol resistance or thegene encoding the cI repressor of the phage L1. This vector can beconstructed using known techniques and those described herein. Presenton the same vector or, alternatively, provided in trans can be genesnecessary to mediate integration of the vector.

The work described herein has resulted in identification of plasmidreplicons which replicate extrachromosomally in mycobacteria (e.g., M.smegmatis, BCG). As described, the pAL5000 replicon has been identified.The same method can be used to identify others which can also be used.

The work described also demonstrates successful construction of E. colimycobacterial shuttle phasmids that are recombinant DNA molecules thatnot only have the ability to replicate in E. coli as plasmids and inmycobacteria as a phages, but also have the ability to be packaged intobacteriophage lambda heads or into mycobacteriophage particles. Itfurther demonstrates that recombinant DNA has been introduced into botha fast-growing mycobacterium (M. smegmatis) and a slow-growingmycobacterium (BCG). This makes it possible to infect M. bovis-BCGvaccine strains with the shuttle phasmids and, thus, to introduce clonedgenes into mycobacteria. Thus, this eliminates the need to develop atransfection system for BCG. That is, because the E. coli-mycobacterialshuttle plasmid, upon transfection into mycobacteria, is packaged intomycobacterial particles, DNA of interest can be introduced stably intoslow-growing mycobacteria (e.g., M. bovis-BCG) by transduction, ratherthan transfection. This makes it possible to produce recombinantmycobacterial vaccine vehicles, which can be used to immunize againstone or more antigens of interest.

The use of in vitro packaging to construct these phasmids can beextended as an efficient strategy for cloning of genes (e.g., genes orDNA of interest encoding an antigen or antigens for one or morepathogens against which an immune response is desired) into thesevectors, as long as the size limits of the packaging system are notexceeded. It is also possible, by screening additional L1::pHC79recombinant phasmids, to determine the maximum amount of DNA that can bedeleted from the L1 phage and to define additional non-essential regionsof the phage genome into which DNA can be inserted.

Construction of Genetically Recombinant Mycobacteria Useful to ExpressDNA of Interest

The method of the present invention is useful to construct a geneticallyrecombinant mycobacterial vehicle for the expression of the protein(s)or polypeptide(s) encoded by DNA of interest incorporated into themycobacterium. Such genetically recombinant mycobacteria have many uses.

Vehicles of the present invention can be used, for example, as vaccinesto induce immunity against the pathogenic antigen encoded by the DNA ofinterest. A pathogen is any virus, microorganism, or other organism orsubstance (e.g., toxins) which causes disease. A vaccine vehicle usefulfor immunizing against leprosy can be made. Because of the extraordinaryadjuvant activity of mycobacteria, such as BCG, such a vaccine would beeffective in producing cell-mediated immunity, particularly of along-term or enduring nature. Genes encoding protein antigens of theleprosy parasite M. leprae have been isolated by Young and are describedin detail in co-pending U.S. patent application Ser. No. 892,095, filedJul. 31, 1986, the teachings of which are incorporated herein byreference. In particular, genes encoding five immunogenic proteinantigens (i.e., antigens of molecular weight 65 kD, 36 kD, 28 kD, 18 kDand 12 kD) have been isolated. In addition, 6 different epitopes encodedby the gene for the 65 kD antigen have been defined. At least one ofthese epitopes has been shown to be unique to M. leprae; the otherepitopes have been shown to be shared with the 65 kD proteins of othermycobacteria.

Through use of the shuttle vectors and recombinant plasmid vectors ofthe present invention, it is possible to introduce into BCG one or moreof the genes encoding M. leprae protein antigens, using methodsdescribed above and in the following examples. The gene encoding the 65kD M. leprae protein has, in fact, been introduced into and expressed byrecombinant BCG. Results of Western blot analysis (FIG. 17) demonstratedthe presence of both the 65 kD M. leprae antigen and the selectablemarker. For example, the gene encoding the 65 kD M. leprae antigen canbe introduced into BCG, stably integrated into its genomic DNA andexpressed at levels sufficient to stimulate or induce an immune responsein a host to which it is administered. As described in detail in Example11, monoclonal antibodies specific for the 65 kD M. leprae protein havebeen used to demonstrate expression of the 65 kD M. leprae gene inextracts of M. smegmatis into which the gene was introduced. Inaddition, the gene encoding the SIV1 envelope protein has been clonedinto a similar plasmid vector, to be introduced into M. smegmatis usingthe techniques described for the Kan and M. leprae genes. A similarconstruct, for introduction into BCG, has also been made. In this way,it is possible to construct a vaccine which is close to ideal, in thatit contains one or more protective antigens of M. leprae, does not havetolerogenic determinants and has an excellent adjuvant for inducingcell-mediated immunity.

In a similar fashion, it is possible to construct a vaccine, using ashuttle or plasmid vector and the method of the present invention, toprovide specific protection against tuberculosis. Such a vaccine isparticularly attractive because of the recently reported finding,described above, that presently-used vaccines are proving to beineffective. Genes encoding immunogenic protein antigens of the tuberclebacillus M. tuberculosis have been isolated and are described inco-pending U.S. patent application Ser. No. 07/010,007, entitled "GenesEncoding Protein Antigens of Mycobacterium Tuberculosis and UsesTherefor", by Robert N. Husson and Richard A. Young, filed Feb. 2, 1987(now abandoned), and in the continuation-in-part application, Ser. No.07/154.331, filed by the Express Mail procedure Feb. 10, 1988), entitled"Genes Encoding Protein Antigens of Mycobacterium Tuberculosis and UsesTherefor", by Robert N. Husson, Richard A. Young and Thomas M. Shinnick,the teachings of which are incorporated herein by reference.

In this case, a gene encoding an immunogenic protein antigen of M.tuberculosis is introduced into BCG by means of a shuttle or plasmidvector, as described above. It is also possible to introduce more thanone M. tuberculosis gene, each encoding a protein antigen, into. BCG.For example, a gene encoding immunogenic M. tuberculosis antigens ofmolecular weight 12 kD, 14 kD, 19 kD, 65 kD and 71 kD, or a combinationof two or more of these genes, can be inserted into BCG, stablyintegrated into genomic DNA and expressed. The result is a vaccine whichis specific for immunization against tuberculosis and which induceslong-lived immunity against the bacillus.

It is also possible, using the method of the present invention, toconstruct a multipurpose or multifunctional vaccine (i.e., a singlevaccine vehicle which contains and expresses DNA of interest whichincludes more than one gene, each gene encoding a protein antigen for adifferent pathogen or toxin). For example, it is possible to introduceinto BCG, using the shuttle vector plasmid or the plasmid vectordescribed, a gene encoding a protein antigen for M. leprae, a geneencoding a protein antigen for M. tuberculosis, a gene encoding aprotein antigen for Leishmania, and a gene encoding a protein antigenfor malaria. Administration of this multi-valent vaccine would result instimulation of an immune response to each antigen and provide long-termprotection against leprosy, tuberculosis, leishmaniasis, and malaria.

The recombinant mycobacteria can also be used as an anti-fertility"vaccine" vehicle. For example, mycobacteria containing DNA encodingantigens such as human gonadotropic hormone (HGH) fragments can be usedas an anti-fertility vaccine and administered as a birth control agent.Vaccine vehicles of the present invention can be used to treat humancancers, such as bladder cancers or melanomas (e.g., by expressinggrowth inhibitors or cytocidal products). In this context, recombinantmycobacteria which contain and express interferon α, β and/or γ, one ormore interleukin (interleukins 1-7) and/or TNF α or β are particularlyuseful. In another application, recombinant mycobacteria can be used toexpress stress proteins, either for the purpose of eliciting aprotective immune response (e.g., against subsequent or long-terminfection) or for the purpose of inducing tolerance in an autoimmunedisease (e.g., rheumatoid arthritis). Stress proteins, such as thosedescribed in co-pending U.S. patent application Ser. No. 207,298,entitled Stress Proteins and Uses Therefore, by Richard A. Young andDouglas Young, filed Jun. 15, 1988, can be used in this purpose. Becauseof their large genomes (e.g., the BCG genome is about 3×10⁶ bp long),mycobacteria can accommodate large amounts of DNA of interest and, thus,can serve as multi-purpose vehicles.

Recombinant mycobacteria of the present invention can be used to producepolypeptide(s) of interest, such as steroids. In this case, all or aportion of a steroid-encoding gene is introduced into an appropriatemycobacterial host, in which it is expressed. Thus, the recombinantmycobacteria provide a valuable means of producing such proteins.

In addition, the shuttle vectors and genetically recombinantmycobacteria of the present invention can be used in a diagnosticcontext. For example, a shuttle plasmid which is specific for (capableof introducing DNA into) a pathogenic organism (e.g., M. tuberculosis,M. avium) and includes DNA encoding a reporter molecule (e.g.,luciferase from a Vibrio bacterium or of firefly origin;β-galactosidase; β-glucoronidase; catechol dehydrogenase) and a strongmycobacterial promoter (the DNA sequence necessary for initiatingtranscription), which controls (drives) expression of the reportermolecule-encoding gene, is constructed. A sample (e.g., blood, urine) isobtained from an individual to be assessed for presence or absence ofthe pathogenic organism. If, for example, the individual is being testedfor tuberculosis, a shuttle plasmid specific for M. tuberculosis isused. The sample is cultured and combined with an appropriate quantityof the M. tuberculosis-specific plasmid. After a short time (e.g.,several hours) under appropriate conditions, the sample is assayed,using known techniques, for the occurrence (presence or absence or ifdesired, the quantity, of the reporter molecule encoded by the DNA inthe vector. If the sample contains M. tuberculosis, even at very lowlevels, the DNA present in the phage will be introduced into theorganism. Once in M. tuberculosis present in the sample, the plasmid(phage) DNA, including that encoding the reporter molecule, will bereplicated. If the reporter molecule is luciferase, a considerablequantity of luciferase will be produced (because production is driven bya strong promoter) and can be detected using standard equipment, such asa photometer. Determination of presence or absence of M. tuberculosisinfection in the individual is thus possible, as is quantitation, ifdesired. Until the present method was developed, available techniques ofdiagnosing tuberculosis were slow (e.g., required several weeks).β-galactosidase, which has now been expressed in mycobacteria, can alsobe used as a reporter molecule.

In any of the uses of the recombinant mycobacteria to express a proteinor polypeptide, it is possible to include in the shuttle vector DNAencoding a signal sequence and, thus, provide a means by which theexpressed protein or polypeptide is made in the cytoplasm and thensecreted at the cell walls. For example, the signal sequence from aantigen, which is secreted in mycobacteria, could be used.Alternatively, the signal sequence for β-galactosidase., agarase or aamylase could be used.

The present invention will now be illustrated by the following examples,which are not to be considered limiting in any way.

EXAMPLE 1

Transfection of M. smegmatis spheroplasts with mycobacteriophage D29 DNA

Spheroplasts of the M. smegmatis strain mc² 6 were prepared according tothe following method. mc² 6 is a single colony isolate that is thepredominant colony type isolated from the ATCC 607 M. smegmatis stockculture. It forms orange rough colonies on regeneration media. Hopwood,D. A. et. al., In: Genetic Manipulation of the Streptomyces-A LaboratoryManual, The John Innes Foundation, Norwich, England (1985).

Spheroplasts of M. smegmatis were prepared as for Streptomyces, usingmedia for spheroplast preparation described by Udou et. al. for M.smegmatis. Udou, T. et al., Journal of Bacteriology, 151: 1035-1039(1982). mc² 6 cells were grown in 40 ml of tryptic soy broth containing1% glucose and 0.2% Tween 80 in a 250-ml baffled-flask at 37° C. withmoderate shaking to an A₆₀₀ -0.2, at which time a 20% glycine solutionwas added to a final concentration of 1%. The cells were incubated foran additional 16 hours and then harvested at room temperature bycentrifuging at 5000×g for 10 minutes. The pellet was washed twice with10 ml of 10.3% sucrose and then resuspended in protoplast (P) buffercontaining 2 mg/ml lysozyme solution. After a 2-hour incubation at 37°C., 5 ml of P buffer was added and the spheroplasts were pelleted bycentrifuging at 3000×g for 7 min. The pellet was resuspended in 10 ml Pbuffer and used within 3 hours.

mc² -11 was isolated as a spontaneous D29-resistant isolate of the ATCC607 M. smegmatis stock culture when 10⁸ cells were mixed with 3×10⁸ D29plaque-forming units and plated on tryptic soy agar plates.D29-resistant colonies arose at a frequency of 10⁻⁷.

mc² 6 spheroplasts were mixed with 1 ug of D29 DNA; one tenth of theresulting mixture was plated on tryptic soy agar plates, with or without0.5M sucrose. They were then overlayed with the appropiate soft agarcontaining 10⁸ mc² 6 cells. The DNase treatment was performed by addingDNase I (Sigma), at a final concentration of 50 ug/ml, to the D29 DNA.

Equivalent amounts of mc² 11 spheroplasts were used in the same manner,but then subsequently overlayed with mc² 6 cells to assay plaque formingunits (pfu).

Phage Plate Stocks: Plate lysates of D29 were prepared on tryptic soyagar media containing 2 mM CaCl₂. M. smegmatis cells that had been grownin a baffled flask at 37° C. in Middlebrook 7H9 broth containing ADCenrichment to midlog phase were mixed with phage diluted in MP buffer(10 mM Tris-HCl, pH 7.6 -10 mM MgCl₂ -100 mM NaCl-2 mM CaCl₂) andincubated at 37° C. for 36 hours, until plates were confluent. The phagewere harvested with MP buffer and then purified on two CsCl equilibriumgradients, followed by extensive dialysis against MP buffer. DNA wasextracted from phage by adding EDTA to a final concentration of 50 mMand treating with proteinase K at 100 ug/ml at 55° C. for 24 hours,followed by phenol-chloroform extraction, and extensive dialysis againstTE buffer.

Transfection: For each transfection, 2.5 ml of the spheroplastsuspension was pelleted in a conical 15-ml polystyrene tube. Thesupernatant fluid was carefully decanted and the spheroplasts wereresuspended in the remaining drop of buffer. After adding 1 ug of DNA ina total volume of less than 10 ul, 0.5 ml of a 25% PEG-1000 (J. T. BakerChemical Co., Phila, Pa.) solution prepared in P buffer was added. Theresulting combination was mixed. Within 3 min, 5 ml of P buffer wasadded to the mixture and the spheroplasts were pelleted as above. Aftercarefully pouring off the supernatant fluid, the pellet was resuspendedin 1 ml of P buffer and samples were transferred to tryptic soy agarwith or without 0.5M sucrose. The plates were then overlayed with 3.0 mlof soft tryptic soy agar and incubated at 37° C. The plaques werecounted after 24 hours of incubation.

EXAMPLE 2

Construction of the shuttle plasmid phAE1

TM4 phage DNA was ligated at a concentration of 250 ug/ml. Aliquots werepartially digested with Sau3A that was serially diluted; fragments thataveraged 30 to 50 kb in length (as analyzed by agarose gelelectrophoresis gel electrophoresis) were obtained in this manner. Thesefragments were ligated at a 1:2 molar ratio of TM4 fragments to pHC79that had been cleaved with BamHI. The packaging of an aliquot of thisligation with in vitro packaging mix (Gigapack plus, Stratagene, SanDiego, Calif.) and subsequent transduction into ER1381 (hsdR mcrA⁺mcrB⁺, E. Raleigh), yielded 10⁶ ampicillin colonies per ug of TM4 DNAinsert, when plated on L agar containing ampicillin at 50 ug/ml.

A pool of 40,000 ampicillin-resistant clones was prepared byhomogenizing colonies in L broth with a glass spreader. Plasmid wasisolated from pools of clones by alkaline-SDS extraction, followed byphenol-chloroform extraction and concentration with ethanol.Covalently-closed plasmid DNA was transfected into mc² 6 spheroplasts asdescribed in Example 1. The plaques were screened for the presence ofpHC79 by performing plaque lifts using the protocol of Benton and Davisand Biotrans nylon membranes (ICN). Benton, W. D. and R. W. Davis,Science, 196: 180-182 (1977). The membranes were hybridized with pHC79DNA that had been nick-translated with ³² P-dCTP and autoradiography wasperformed.

EXAMPLE 3

Infection of BCG and M. smegmatis with shuttle plasmid phAE1

BCG-Glaxo (W. Jones) was propagated in Middlebrook 7H9 broth (Difco)containing ADC enrichment (Difco) and 0.5% Tween 80 (Sigma) in standingcultures at 37° C. Lawns of BCG-Glaxo or mc² 6 cells were prepared bymixing 10⁸ BCG-cells with supplemented top soft agar and pouring onDubos agar without Tween 80 (Gibco) supplemented with OADS enrichment(Difco). Jones, W. D., Jr., Tubercle, 60: 55-58 (1979). The 4 phages,DS6A, TM4, phAE1, and 33D were serially diluted and spotted on the twolawns. The plates were read at 14 days and 2 days for BCG-Glaxo and M.smegmatis, respectively.

EXAMPLE 4

Cloning of aminoglycoside phoshotransferase gene into phAEI

A 1.6 kb EcoRI fragment encoding the aminoglycoside phosphotransferasegene (aph) from Tn903 was cloned into phAE1 by taking advantage ofcosmid cloning strategy. Plasmid phAE1 DNA was isolated from E. coli andcut with EcoRI, the 1.6 kb fragment was ligated to these large DNAmolecules. The ligation product was packaged into phage lambda in vitro,yielding particles which transduced kanamycin-resistance andampicillin-resistance to E. coli cells. Plasmid DNA was isolated fromthese E. coli cells and shown to yield high frequencies ofplaque-forming units when transfected into M. smegmatis mc² 6protoplasts. This demonstrates that it is possible to clone at least 1.6kb of additional DNA into the unique EcoRI site of phAE1. Similarresults were obtained with the shuttle plasmid phAE2, a shuttle vectorwhich has similar characteristics to those of phAE1 but is 2 kb smallerin size than phAE1, which should allow for the cloning of at least 3.6kb of additional DNA. In both cases, introduction of the aph generesulted in introduction of a new NruI site, providing proof thatadditional DNA fragments can be cloned and stably maintained in theshuttle phasmids. Thus, these vectors without further modification canbe useful for cloning additional genes into mycobacteria.

EXAMPLE 5

Stable expression of a selectable marker in mycobacteria using a shuttleplasmid

Shuttle phasmids were constructed from the phage L1 (ATCC #27199) in amanner similar to those constructed for the TM4 phage. Doke, S.,Kumamoto Medical Journal, 34:1360-1373 (1960). All of the L1-shuttlephasmids identified have the ability to lysogenize M. smegmatis. L1 hasbeen shown to integrate into M. smegmatis chromosomal material and toform stable lysogens. Other phage, such as L3 (ATCC #27200), a phagewhich remains as a plasmid (extrachromosomal) and L5 (ATCC #27201) canalso be used in constructing shuttle phasmids. Results showed that theseshuttle phasmids will lysogenize M. smegmatis and thus made it possibleto stably integrate DNA of interest into mycobacteria for the firsttime. The aph gene was cloned into the unique EcoRI site of theL1-shuttle plasmid designated phAE15, as described above for theTM4-shuttle phasmids in E. coli. M. smegmatis cells (mc² 6) wereoverlayed on top of agar on a Dubos agar plate containing kanamycin.Dilutions of the shuttle plasmid phAE15 and phAE19 (phAE15 with theclone aph gene) were spotted on the agar lawn. The plate was incubated 5days at 37° C. for 5 days. The colonies that grew all had beenlysogenized with the L1-shuttle plasmid into which the aph gene had beencloned. The resulting shuttle plasmid, phAE19, was able to lysogenize M.smegmatis cells. The resulting lysogens expressed the cloned aph genebecause they were resistant to kanamycin. Furthermore, these lysogensyielded mycobacteriophage particles that also expressed thekanamycin-resistant phenotype upon subsequent transfer andlysogenization of kanamycin-sensitive M. smegmatis cells. Transfer ofthese phages results in cotransduction of the lysogenic state (i.e.immunity to superinfection) and kanamycin resistance. The L1 phage, usedto lysogenize M. smegmatis, does not plaque on BCG. However, variants ofboth L1 and the shuttle plasmid phAE19 which do form placques on BCGhave been isolated. These can be tested for their ability to introduceand stably express genes of interest in BCG and M. tuberculosis by meansof temperate shuttle phasmids. Thus, these phages have the ability tostably introduce DNA of interest into M. smegmatis. In addition, hostrange variants (e.g., phAE19) which will infect and lysogenize BCG havebeen isolated. This has made it possible to produce a recombinantmycobacterium, containing DNA of interest. Such recombinant mycobacteriacan be used as a vaccine.

EXAMPLE 6

Integration of mycobacteriophage L1 and L1-shuttle plasmid DNA into theM. smegmatis chromosome

Phage L1 was obtained by plating the culture supernate from anunspeciated Mycobacterium, ATCC 27199, grown in tryptic soy brothcontaining 0.05% Tween 80, on cloned M. smegmatis strain, mc² 6. Jacobs,W. R. Jr., Tuckman, M. & Bloom, B. R. Nature, 327:532-535 (1987). Thephage was plaque-purified and high-titered plate lysates were obtainedfrom mc² 6 grown on Dubos agar medium (without Tween) containing 2 mMCaCl₂ at 37° C. Phage particles were purified by CsClequilibrium-density centrifugation and phage DNA was isolated asdescribed previously. Jacobs, W. R. Jr., Tuckman, M & Bloom, B. R.Nature, 327:532-535 (1987). L1-shuttle phasmids were constructedfollowing the previously described protocol using pHC79 as the cosmidand the substitution of L1 DNA for TM4 DNA. Jacobs, W. R. Jr., Tuckman,M. & Bloom, B. R. Nature, 327:532-535 (1987). The aph gene from Tn903was introduced into one L1 -shuttle plasmid, phAE154, by ligating phAE15DNA cleaved at the unique EcoRI site to the Tn903 EcoRI aph cassette(Pharmacia). The resulting ligation was packaged in vivo intolambda-phage heads, which were then transduced into the E. coli strain2338, selecting for both ampicillin- and kanamycin-resistance. Jacobs,W. R. et al. Proc. Natl. Acad. Sci. U.S.A., 83: 1926-1930 (1986).Plasmid DNA was isolated from the E. coli, transfected into mc² 6protoplasts and the resulting mycobacteriophage was designated phAE19.Lysogens were purified from turbid plaques arising after spottingphasmids on agar containing mc² 6 cells. Putative lysogens were testedfor release of phages and resistance to superinfection by L1.Chromosomal DNA was isolated using a Braun homogenizer, followed byphenol-chloroform extractions. The Southern analysis was performed usingBiotrans (ICN) nylon membranes following the manufacture'srecommendations. L1 DNA was radiolabelled using a nick translation kit(BRL) and χ-³² P!-dCTP (Amersham).

EXAMPLE 7

Expression of kanamycin-resistance by lysogeny using the temperateshuttle plasmid phAE19

M. smegmatis, mc² 6, 2×10⁷ ! cells, grown in shaking cultures at 37° C.in Middlebrook 7H9 broth supplemented with ADC enrichment and 0.05%Tween 80 (M-ADC-TW broth), were mixed with 3 ml Dubos top agar andoverlayed onto a Dubos agar plate containing 15 ug/ml kanamycin. Lysatesof the L1-shuttle phasmids, phAE15 and phAE19 (-phAE15::aph), werefiltered through a 0.45 um filter and diluted to approximately 5×10⁶pfu/ml using MP buffer. Jacobs, W. R. Jr., Tuckman, M. & Bloom, B. R.Nature, 327: 532-535 (1987). Serial tenfold dilutions (10 ul) werespotted in the designated areas, and the plates were incubated for 5days at 37° C. As shown in FIG. 7, colonies appeared where phAE19lysogenized mc² 6 cells, thus demonstrating expression ofkanamycin-resistance. In multiple experiments, kanamycin-resistancecolonies were not observed from either spontaneous mutants of mc² 6cells or mc² 6 cells lysogenized with phAE15. The M. smegmatis strain,designated mc² 96, which is mc² 6 lysogenized with phAE19 was depositedJul. 22, 1988. at the American Type Culture Collection (Rockville, Md.)under Accession No. 67746. All restrictions on public access to thedeposit will be removed irrevocably upon grant of a United States patentbased on this application.

EXAMPLE 8

Construction and analysis of E. coli-mycobacteria shuttle plasmids

Plasmid pAL5000 DNA, isolated as described previously, was partiallydigested with MboI and linear fragments of 5 kb were isolated from anagarose gel following electrophoresis. Birnboim, H. & Doly, J. NucleicAcid Res., 7:1513-1525 (1979). These fragments were ligated to thepositive selection vector pIJ666, which contain the neo gene originatingfrom Tn5, and the P15A origin Of replication and cat gene from pACYC184,that had been cleaved with BamHI and EcoRV and transformed into E. coli.Kieser, T. and R. E. Melton, Gene, 65:83-91 (1988); Berg, D. E. et al.,Proc. Natl. Acad. Sci. U.S.A., 72:3628-3632 (1975); Chang, A. C. Y. andS. N. Cohen, J. Bact., 134:1141-1156 (1978) and Chi, T. et al, J. Bact.,133:816-821 (1978). Chloramphenicol-resistant transformants (200colonies, resistant to 25 g/ml) were pooled and grown in mixed culture,from which plasmids were isolated. Birnboim, H. and J. Doly, NucleicAcid Res., 7:1513-1525 (1979). This library of pIJ666::pAL5000 hybridplasmids was transformed into M. smegmatis by electroporation using theGene Pulser (Biorad) electroporator. Chassy, B. M. and T. L. FlickingerFEMS Microbiology Letters, 44:173-177 (1987). Fresh cultures of mc² 6cells were grown in M-ADC-TW broth with shaking to an A₆₀₀ -1.7. Thecells were harvested by centrifugation, washed in electroporation buffer(7 mM phosphage, pH7.2-272 mM sucrose) and resuspended to one tenth theoriginal volume. Plasmid DNA (1 ug) was added to an electroporationcuvette containing 0.8 ml of M. smegmatis cells. Following a 10 minuteincubation on ice, the cells were subjected to a single pulse ofelectroporation (25 uF at 6250 V/cm), then mixed with an equal volume ofM-ADC-TW broth and incubated at 37° C. for 2 hours. The cells were thenplated on 7H10 agar plates containing 10 ug/ml kanamycin and incubatedfor 7 days at 37° C. The kanamycin-resistant transformants weresubcultured in 7H9-ADC-TW both containing 10 ug/ml kanamycin andretained their ability to plaque phage D29, confirming that they were M.smegmatis. Froman, S. et al., Am. J. Public Health, 44:1326-1334 (1954).These transformants were also resistant to 100 ug/ml of chloramphenicol.Plasmid DNA was isolated from 1 ml sample of cells by a modification ofthe procedure of Birnboim and Doly, incubating overnight sequentially inlysozyme, alkaline-SDS and finally high-salt. The DNA isolated from M.smegmatis was transformed into 2338 and yielded more than 10⁴kanamycin-resistant E. coli transformants per ug of DNA. Birnboim, H.and J. Doly, Nucleic Acids Res., 7:1513-1525 (1979). All unique plasmidsisolated from individual E. coli transformants could transform andconfer kanamycin- and chloramphenicol-resistance to M. smegmatis.

EXAMPLE 9

Transformation of M. smegmatis and BCG with shuttle plasmid DNA

The BCG-Pasteur substrain P1173P2 was grown in M-ADC-TW broth shaking at37° C. for 5 days (estimated viability 4.5×10⁷ cfu/ml). These cells weretransformed by electroporation with the pIJ666:pAL5000 recombinantlibrary following the same procedure described above and plated on 7H10agar containing ADC enrichment and 20 ug/ml of kanamycin. A pool of 45kanamycin-resistant BCG cells was cultured in liquid medium containing20 ug/ml of kanamycin for 3 weeks at 37° C. From this culture, plasmidswere isolated as described in Example 8. They were all 11.2 kb in sizeand conferred kanamycin-resistance upon E. coli cells when transformed.This plasmid DNA was again used to transform BCG cells. The plates shownabove were incubated for 18 days at 37° C. and then photographed.BCG-Pasteur substrain transformed with a shuttle plasmid, designatedpYUP1100 (also referred to or designated pYUB13), which includes thegene encoding kanamycin resistance and the gene encoding chloramphenicolresistance, was deposited Jul. 22, 1988 at the American Type CultureCollection (Rockville, Md.) under Accession No. 67745. All restrictionson public access to the deposit will be removed irrevocably upon grantof a United States patent based on this application.

The M. leprae gene encoding stress-induced 65 kDa antigen has also beenintroduced and expressed in M. smegmatis and BCG. The M. leprae gene wascloned into an E. coli-Mycobacteria shuttle plasmid, designated pYUB12,which is a member of the group of shuttle plasmids, previouslydesignated pYUP, which includes pYUP1100. The resulting construct,pYUB39, was transformed into both M. smegmatis and BCG-Pasteur and celllysates from transformants were electrophoresed on SDS-polyacrylamidegels. The resulting gel was blotted onto nylon membrane that was thenprobed with a mouse monoclonal antibody that recognizes the M.leprae-specific epitope IIE9. The blot was then probed withmouse-specific rabbit antibodies linked to alkaline phosphatase,developed for phosphatase activity, and photographed. The resulting geldemonstrates that the cloned gene encoding the foreign M. leprae 65 kDaantigen is expressed in both M. smegmatis and BCG, as represented inFIG. 17, which is a photograph of the Western blot analysis of the SDSpolyacrylamide gel electrophoresis of cell lysates containing therecombinant plasmids pYUB12 or pYUB39.

EXAMPLE 10

Construction of a recombinant plasmid for introduction of the Kan geneinto M. smegmatis and integration of Kan into M. smegmatis genome

The following bacterial strains were used: RY1103 (DB6507, Bach, M. L.et al., Proceedings of the National Academy of Sciences, USA, 76:386-390(1979)) HB101,pyrF::Tn5,thr-,leu-,pro-,B1-,r-,m-,suII and RY1107(DB6566, Rose, M. et al., Gene, 29:113-124 (1984))B15,pyrF::Mu,trp_(am),lacZ_(am),hsdR⁻,m⁺,Su³¹. Both were obtained fromDr. David Botstein (Massachusetts Institute of Technology).Y1109(DH5alpha)F⁻,endA1,hsdR17(rK,mK⁺), supE44, thi1, recA1, gyrA96,rela, del (argF-lacZYA) U169, lambda⁻,phi80dlacZde1M15, which wasobtained from Bethesda Research Laboratories. MC² -6, a single colonyisolate M. smegmatis prototroph, which was obtained from Dr. WilliamJacobs (Albert Einstein College of Medicine). FOA^(R) -3 is aspontaneous mutant of MC² -6 to uracil auxotrophy and resistance to5-fluoro-orotic acid. M. bovis-BCG (Moreau) is ATCC 35736. M. bovis-BCG(Montreal) is ATCC 35735.

Mycobacterial Genomic DNA Libraries

M. smegmatis genomic DNA was obtained from MC² -6 after growth intryptic soy broth supplemented with glucose and Tween 80. Cultures weregrown to saturation with glycine added to 0.5% for the last severalhours. Cells were harvested by centrifugation, washed and resuspended in50 mM Tris pH8.0, 10 mM EDTA, 10% sucrose and then treated with 0.2 mg.per ml. lysozyme for one hour, followed by 50 mM EDTA and 1% SDS for 15minutes. Multiple phenol:chloroform extractions were performed, followedby isopropanol precipitation, RNAse treatment, phenol:chloroformextraction, chloroform extraction and ethanol precipitation. The pelletswere washed with 70% ethanol and resuspended in TE pH7.5. M. bovis-BCG(Moreau) genomic DNA was a generous gift of Dr. Graskinsky.

Mycobacterial genomic DNA was partially digested with Sau3A, sizeselected by agarose gel electrophoresis onto DE81 paper, eluted withhigh salt, ethanol precipitated and ligated into pUC19 which had beencleaved with BamHI and treated with calf intestinal phosphatase.DH5alpha, made competent for transformation by the procedure of Hanahan,were transformed with this ligation and plated onto Luria Bertani agarcontaining 50 ug/ml ampicillin. The proportion of colonies containingrecombinant plasmids was determined by plating onto indicator platescontaining XGa1 and IPTG and determining the ratio of white colonies tototal (white plus blue) colonies. Pooled plasmid DNA was obtained byscraping colonies from the plates, resuspending in 50 mM Tris pH 8.0, 10mM EDTA, 50 mM glucose. The resulting suspension was processed by thealkaline lysis method for obtaining plasmid DNA. The M. smegmatisrecombinant DNA library consists of 35,000 independent initialtransformants, of which 85% were recombinant. The M. bovis-BCGrecombinant DNA library consists of 64,000 independent initialtransformants of which 55% were recombinant.

Isolation of recombinant plasmids containing the mycrobacterial pyrFgene

Y1103 and Y1107 were made competent by the method of Hanahan,transformed with the plasmid library DNA and plated on minimal agarplates. Of 180,000 transformants initially screened for the M. smegmatislibrary, 31 were able to grow on minimal medium.

Plasmid DNA Isolation, Restriction Mapping and DNA Sequencing

Plasmid DNA was isolated from liquid cultures by the alkaline lysismethod. Restriction mapping of recombinant plasmid DNA was performedwith multiple enzymes using standard methods. DNA sequencing wasperformed using the dideoxy method after subcloning into M13mp18 andM13mp19, using sequencing kits from New England Biolabs and U.S.Biochemicals.

EXAMPLE 11

Integration of the M. leprae 65 KD gene into M. smegmatis genomic DNA

Construction of recombinant plasmids expressing kanamycin resistance andthe M. leprae 65 kD antigen

pPP25, a recombinant plasmid containing DNA from M. smegmatis able tocomplement pyrF⁻ E. coli, was digested with BamHI and ligated to the 1.3kB BamHI fragment encoding aminoglycoside phosphotransferase of Tn903,isolated from pUC4kSAC. The Eco RI fragment of Y3178 containing the geneencoding the M. leprae 65 kD antigen was subsequently cloned into theunique XhoI and EcoRV sites in the mycobacterial DNA in this plasmid. Ineach case the transcriptional orientations of the mycobacterial openreading frame, the kanamycin resistance gene and the M. leprae 65 kDgene were determined to be in the same orientation.

Transformation of Mycobacteria by Electroporation

M. smegmatis and M. bovis-BCG were grown in Middlebrook 7H9 mediumsupplemented with ADC enrichment and 0.05% Tween 80 (M-ADC-Tw) to anA₆₀₀ of approximately 0.3 to 0.5. Cells were harvested bycentrifugation, washed in 10 mM Hepes pH7.0, centrifuged and resuspendedin 1/10 volume 10 mM Hepes pH7.0, 10% glycerol (M. smegmatis), or washedand resuspended in 1/10 volume 7 mM sodium phosphate pH 7.2, 272 mMsucrose (BCG). DNA was added and the cells were exposed to a singlepulse of 6.25 kV/cm at 25 microfarads using the Biorad Gene Pulser.Three to five volumes of M-ADC-Tw were then added, the cells wereincubated for 2-3 hours at 37° C., centrifuged, resuspended in a smallvolume of M-ADC-Tw and plated on tryptic soy agar supplemented with 1%glucose, containing 10 ug/ml kanamycin (M. smegmatis) or Middlebrook7H10 agar supplemented with ADC enrichment containing 10 ug/mlkanamycin.

Southern Blot Analysis

Genomic DNA from mycobacterial transformants was digested withrestriction enzymes, electrophoresed in agarose gels, transferred tonitrocellulose and probed with DNA labelled with ³² P by nicktranslation, all using standard procedures.

Western Blot (Immunoblot) Analysis

Expression of the 65 kD M. leprae protein was demonstrated using Westernblot techniques. Lysates of mycobacterial and E. coli transformants weresubjected to SDS polyacrylamide gel electrophoresis, electro-transferredto nitrocellulose, and probed with the monoclonal antibody IIIE9 at adilution of approximately 1:1000 using standard techniques. TheProtoblot kit (Promega Biotec) was used to detect binding of theantibody and was used according to the manufacturer's instructions. Thisresulted in detection of expression of the 65 kD protein in cellstransformed with the plasmid containing the M. leprae gene.

Equivalents

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

We claim:
 1. A recombinant mycobacterium capable of expressing DNAencoding a selectable marker incorporated therein, wherein the DNAencoding the selectable marker is stably integrated into genomic DNA ofthe recombinant mycobacterium.
 2. The recombinant mycobacterium of claim1, which is selected from the group consisting of:a. Mycobacteriumsmegatis; b. Mycobacterium bovis-BCG; c. Mycobacterium avium; d.Mycobacterium phlei; e. Mycobacterium fortuitum; f. Mycobacterium lufu;g. Mycobacterium tuberculosis; h. Mycobacterium habana; i. Mycobacteriumscrofulaceum; j. Mycobacterium intracellare; and k. any genetic variantsthereof.
 3. The mycobacterium of claim 1, wherein the DNA encoding theselectable marker is a gene encoding antibiotic resistance.
 4. Themycobacterium of claim 3, wherein the antibiotic is selected from thegroup consisting of kanamycin, viomycin, thiostrepton, hygromycin andbleomycin.
 5. The mycobacterium of claim 4, wherein the antibiotic iskanamycin.
 6. A recombinant Mycobacterium tuberculosis capable ofexpressing DNA encoding a selectable marker incorporated therein,wherein the DNA encoding the selectable marker is stably integrated intogenomic DNA of the recombinant Mycobacterium tuberculosis.
 7. Themycobacterium of claim 6, wherein the DNA encoding the selectable markeris a gene encoding antibiotic resistance.
 8. The mycobacterium of claim7, wherein the antibiotic is selected from the group consisting ofkanamycin, viomycin, thiostrepton, hygromycin and bleomycin.
 9. Themycobacterium of claim 8, wherein the antibiotic is kanamycin.
 10. Arecombinant Mycobacterium bovis-BCG capable of expressing DNA encoding aselectable marker incorporated therein, wherein the DNA encoding theselectable marker is stably integrated into genomic DNA of therecombinant Mycobacterium bovis-BCG.
 11. The mycobacterium of claim 10,wherein the DNA encoding the selectable marker is a gene encodingantibiotic resistance.
 12. The mycobacterium of claim 11, wherein theantibiotic is selected from the group consisting of kanamycin, viomycin,thiostrepton, hygromycin and bleomycin.
 13. The mycobacterium of claim12, wherein the antibiotic is kanamycin.
 14. The mycobacterium of claim1, wherein an endogenous mycobacterial gene is replaced by the DNAencoding the selectable marker.
 15. The mycobacterium of claim 1,wherein an endogenous mycobacterial gene is rendered nonfunctional bythe DNA encoding the selectable marker.
 16. The mycobacterium of claim6, wherein an endogenous mycobacterial gene is replaced by the DNAencoding the selectable marker.
 17. The mycobacterium of claim 6,wherein an endogenous mycobacterial gene is rendered nonfunctional bythe DNA encoding the selectable marker.
 18. The mycobacterium of claim10, wherein an endogenous mycobacterial gene is replaced by the DNAencoding the selectable marker.
 19. The mycobacterium of claim 10,wherein an endogenous mycobacterial gene is rendered nonfunctional bythe DNA encoding the selectable marker.